WO2024102739A2 - Production de virus adéno-associé (aav) - Google Patents

Production de virus adéno-associé (aav) Download PDF

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WO2024102739A2
WO2024102739A2 PCT/US2023/078957 US2023078957W WO2024102739A2 WO 2024102739 A2 WO2024102739 A2 WO 2024102739A2 US 2023078957 W US2023078957 W US 2023078957W WO 2024102739 A2 WO2024102739 A2 WO 2024102739A2
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seq
mutations
enzyme
identity
nucleotide sequence
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WO2024102739A3 (fr
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Joseph J. HIGGINS
Ray TABIBIZAR
Feng Yao
Quan Karen ZHU
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Saliogen Therapeutics Inc
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Saliogen Therapeutics Inc
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates, in part, to a method of making a viral particle packaging and producer stable cell line using transposon(s) encoding genes required for AAV production and an enzyme capable of performing targeted genomic integration.
  • CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No.63/432,220, filed November 7, 2022, which is incorporated by reference herein in its entirety.
  • SEQUENCE LISTING The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety.
  • AAV Adeno-associated virus
  • the genome includes three open reading frames (ORFs) encoding for replication (non-structural) proteins (rep), capsid (structural) proteins (cap), and the assembly activating protein (AAP). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. See Naso et al., BioDrugs.2017;31(4):317-334.
  • the coding sequences for the rep, cap, and AAP genes are flanked by inverted terminal repeats (ITRs). The 145-nt ITRs are partially paired, and they fold upon themselves to maximize base pairing and form a T-shaped hairpin structure.
  • the rep gene encodes four Rep proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging.
  • the cap gene expression results in viral capsid (Cap) proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. Naso et al. (2017).
  • the AAV genome contains two viral promoters known as p5 and p19, which regulate the transcription of the four Rep proteins with molecular masses of 78, 68, 52, and 40 kDa (Rep68 and Rep40 being the splice variants of Rep78 and Rep52, respectively).
  • cap gene is driven by the P40 promoter and regulated by alternative splicing and different translation initiation sites, resulting in three cap proteins (VP1, VP2, and VP3) that form an icosahedral capsid of 3.9 kDa.
  • the molecular ratio of these proteins (VP1:VP2:VP3) is approximately 1:1:10.
  • the AAV genome also encodes for the AAP in an alternative ORF of the cap gene that plays a major role for capsid assembly. See Penaud-Budloo et al., Mol Ther Methods Clin Dev 2018;8:166-80.
  • AAV includes 12 different AAV serotypes.
  • AAV belongs to the genus Dependoparvovirus (the Parvoviridae family) because it needs the presence of a helper virus for replication and assembly, e.g., adeno-, herpes-, human papilloma- or vaccinia viruses.
  • a helper virus for replication and assembly e.g., adeno-, herpes-, human papilloma- or vaccinia viruses.
  • the virus gets internalized by endocytosis.
  • the viral capsid sheds to release the single-stranded AAV genome which is then converted to double-stranded DNA.
  • the free end of the ITR hairpin hereby acts as a primer for the DNA synthesis.
  • AAV can infect dividing or non-dividing cells.
  • AAV derived vectors have several advantages for viral-based gene therapy. AAV is believed to be non-pathogenic for humans, which makes it a suitable vehicle for delivery of genetic material to human cells. AAV also has low cytotoxicity and elicits a very mild immune response. Therefore, AAV vectors are well suitable for in vivo gene delivery. Furthermore, different AAV serotypes can target various tissues. Due to their properties, AAV derived vectors are becoming a more preferable tool as compared to adenovirus- and retrovirus-derived vectors.
  • the present application provides methods of making viral particle packaging and producer cell lines.
  • the methods comprise transfecting a cell with two or more nucleic acids.
  • the nucleic acids encode one or more of: (a) an enzyme capable of performing targeted genomic integration, (b) an inducible viral replication (Rep) gene, (c) an inducible viral Capsid (cap) gene, (d) one or more adenoviral auxiliary genes (e.g., selected from one or more of E1A, E1B, E4, E2A, and VA of an AAV), (e) an insulator (e.g., selected from HS4, D4Z4), (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV inverted terminal repeats (ITRs).
  • an enzyme capable of performing targeted genomic integration e.g., an enzyme capable of performing targeted genomic integration
  • Rep inducible viral replication
  • Cap inducible viral Capsid
  • adenoviral auxiliary genes e.g., selected from one or more of E1A, E1B, E4, E2A, and VA of an AAV
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the resulting transfected cell expresses the transgene in association with the viral particle.
  • the cell is transfected with two nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV ITRs.
  • the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV ITRs.
  • the cell is transfected with three nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, and (f) one or more terminal ends recognized by the enzyme; and the third nucleic acid encodes (g) a transgene flanked by AAV ITRs.
  • the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration
  • the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, and (f) one or more terminal ends recognized by the enzyme
  • the third nucleic acid encodes (g) a transgene flanked by AAV ITRs.
  • the nucleic acids encode one or more of: (a) an enzyme capable of performing targeted genomic integration, (b) an inducible viral replication (Rep) gene, (c) an inducible viral Capsid (cap) gene, (d) an insulator (e.g., selected from HS4, D4Z4), (e) one or more terminal ends recognized by the enzyme, and (f) a transgene flanked by AAV inverted terminal repeats (ITRs).
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the resulting transfected cell expresses the transgene in association with the viral particle.
  • the E1A, E1B+ cell is transfected with two nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) an insulator, (e) one or more terminal ends recognized by the enzyme, and (f) a transgene flanked by AAV ITRs.
  • the cell is transfected with three nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) an insulator, and (e) one or more terminal ends recognized by the enzyme; and the third nucleic acid encodes (f) a transgene flanked by AAV ITRs.
  • the cell is transfected with four nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (d) an insulator, and (e) one or more terminal ends recognized by the enzyme; the third nucleic acid encodes (c) an inducible viral cap gene; and the fourth nucleic acid encodes (f) a transgene flanked by AAV ITRs.
  • AAV adeno-associated virus
  • the methods make use of a technique to include, among other elements, the AAV viral replication (Rep) and Capsid (Cap) genes under a control of an inducible promoter, in an E1A, E1B+ cell.
  • the inducible Rep and Cap genes (under a control of an inducible promoter), AAV vector DNA sequences, and essential helper genes are included in a single genetic construct such as, e.g., a donor plasmid flanked by end sequences recognized by an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme in accordance with embodiments of the present disclosure).
  • a transgene of interest can be encoded by a separate nucleic acid, or it can be part of the genetic construct encoding the inducible Rep and Cap genes, AAV vector DNA sequences, and essential helper genes.
  • methods for making a packaging and producer cell line with a dual donor/helper system are provided.
  • inducible Rep and Cap genes (sometimes collectively referred to as “Rep/Cap genes”), and a transgene of interest are encoded by respective separate nucleic acids.
  • the dual donor/helper system includes, without limitation, a nucleic acid encoding inducible Rep and Cap genes, a nucleic acid (e.g., a donor expression vector, such as a helper AAV donor plasmid) encoding a transgene of interest, and a nucleic acid encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme).
  • a nucleic acid e.g., a donor expression vector, such as a helper AAV donor plasmid
  • an enzyme capable of performing targeted genomic integration e.g., without limitation, a mobile element enzyme
  • the present application provides a method of making a viral particle packaging and producer cell line, the method comprising transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, a second nucleic acid encoding a donor DNA comprising inducible viral replication (Rep) and Capsid (cap) genes, adenoviral auxiliary genes selected from E1A and E1B, and insulators (optionally selected from HS4, D4Z4), and comprising terminal ends recognized by the enzyme, and a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs), to thereby result in a transfected cell that expresses the transgene in association with the viral particle.
  • ITRs inverted terminal repeats
  • a method of making a viral particle packaging and producer cell line comprises transfecting an E1A, E1B+ cell with: a first nucleic acid encoding an enzyme capable of performing targeted genomic integration; a second nucleic acid encoding a donor DNA comprising inducible viral replication (Rep) and Capsid (Cap) genes, adenoviral auxiliary genes selected from E4ORF6, E2A and VA RNA, and insulators (e.g., without limitation, HS4 and/or D4Z4), and comprising terminal ends recognized by the enzyme; wherein the inducible expression of AAV viral replication (Rep) and Capsid (cap) genes, as well as the adenoviral auxiliary genes E4ORF6 and E2A, are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof, and a third nucleic acid encoding an enzyme capable of performing targeted genomic integration
  • inducible Rep/Cap genes and a nucleic acid e.g., a donor expression vector, such as a helper AAV donor plasmid
  • a nucleic acid e.g., a donor expression vector, such as a helper AAV donor plasmid
  • the single donor/helper system includes, without limitation, a nucleic acid (e.g., a donor expression vector) encoding inducible Rep and Cap genes and a transgene of interest, and a nucleic acid encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme).
  • a method of making a viral particle packaging and producer cell line comprises transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration; and a second nucleic acid encoding viral packaging genes, viral helper genes, insulators (e.g., without limitation, HS4 and D4Z4), and a transgene, and comprising terminal ends recognized by the enzyme, to thereby result in a transfected cell that expresses the transgene in association with a viral particle.
  • insulators e.g., without limitation, HS4 and D4Z4
  • the E1A, E1B+ cell is, without limitation, a HEK293, E1A, E1B+ engineered CHO-K1, or Sf9 cell line.
  • the E1A, E1B+ cell is, without limitation, a Chinese hamster ovary (CHO), baby hamster kidney (BHK), human embryonic kidney (HEK293T) cells, Vero cell, or Spodoptera frugiperda 9 (Sf9) cell
  • the viral helper genes comprise adenoviral auxiliary genes.
  • the adenoviral auxiliary genes are selected from one or more of E1A, E1B, E4ORF6, E2A, and VA of an AAV, optionally E1A and E1B.
  • the viral packaging genes comprise rep and cap genes of viral replication (Rep) and Capsid (Cap) proteins of an AAV.
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO- containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the tetO-containing AAV2 P40 promoter comprises a nucleotide sequence of SEQ ID NO: 816, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the P40 intron is an AAV2 P40 intron.
  • the AAV2 P40 intron comprises a C276A substitution, or a substitution at position corresponding thereto relative to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises one or more mutated translation start sites (ATGs), optionally wherein the translation start sites are mutated to one of CTG, ACG, and TTG.
  • the AAV2 P40 intron comprises substitutions at one or more positions A13, A32, T42, A61, A71, A89, A203, A246, A258, and T282, or one or more positions corresponding thereto, relative to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises substitutions at one or more positions A13C, A32C, T42C, A61C, A71T, A89C, A203T, A246C, A258C, and T282C corresponding to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 818, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 819, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 817, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified TATA box element has the nucleotide sequence of TATATAA.
  • the tetO- containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 823, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the tetO-containing P40 promoter is a tetO-containing AAV2 P40 promoter.
  • the AAV2 P40 promoter comprises the nucleotide sequence of SEQ ID NO: 820, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified P40 intron is a AAV9 P40 intron.
  • the modified AAV9 P40 intron comprises the nucleotide sequence of SEQ ID NO: 821, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified TATA box element has the nucleotide sequence of TATATAA.
  • the tetO-containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV9 P40 intron comprises the nucleotide sequence of SEQ ID NO: 822, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the tetO-containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV9 P40 intron comprises a nucleotide sequence of SEQ ID NO: 824, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the P40 promoter further comprises one or more enhancer elements.
  • the enhancer element comprises one or more cis-acting elements, optionally selected from an Sp1 binding site, GC rich sequence, GCGGAAC motif, TAATGARAT element, AP1 binding site, and CCAAT box element.
  • the enhancer element comprises about 1 to about 5 Sp1 binding sites, optionally about 1, or about 2, or about 3, or about 4, or about 5 Sp1 binding sites.
  • the enhancer element comprises about 1 or about 2 GC rich sequences.
  • the enhancer element is derived from an hCMV Enhancer Element-3.
  • the hCMV Enhancer Element-3 comprises the nucleotide sequence of SEQ ID NO: 830 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the expression of the Rep and Cap proteins is controlled by an inducible promoter, optionally an antibiotic-dependent promoter.
  • the antibiotic-dependent promoter is tetracycline- or a variant thereof dependent promoter and a tetracycline-repressor-based (t-REx) system is used.
  • the antibiotic- dependent promoter is tetracycline- or a variant thereof dependent promoter and a tetracycline-controlled transactivator (rtTA) system is used.
  • the antibiotic-dependent promoter is a coumermycin/novobiocin promoter, or a variant thereof.
  • the viral particle is an AAV, and wherein the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.
  • any one or more of the first, second, and third nucleic acids is encoded by a single nucleic acid.
  • the second nucleic acid and/or the third nucleic acid are included in a single expression vector.
  • the first and second nucleic acids are included in a single expression vector
  • the third nucleic acid is included in an expression vector that is different from the expression vector including the first and second nucleic acids.
  • the single expression vector comprises a plasmid.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 825 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 826 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 841 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 842, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 843, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • one or more of the first, second, and third nucleic acids is RNA, optionally mRNA, optionally synthetic mRNA. In embodiments, one or more of the first, second, and third nucleic acids is DNA, optionally plasmid DNA. In embodiments, one or more of the first, second, and third nucleic acids is an expression vector, wherein the expression vector is optionally a plasmid.
  • the transfected cell generates using the method of making a viral particle packaging and producer cell line in accordance with embodiments of the present disclosure comprises a donor DNA comprising the transgene flanked by AAV inverted terminal repeats (ITRs).
  • the terminal ends or ITRs comprise the nucleotide sequence of SEQ ID NO: 831 and/or SEQ ID NO: 832, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the transgene has a size of about 200,000 base pairs or less.
  • the methods in accordance with the present disclosure can be used to produce viral particles carrying a transgene of a large size.
  • the transgene has a size of at least 200,000 base pairs.
  • the transgene has a size of about 200,000 base pairs.
  • the method further comprises culturing the transfected cell in a medium that expands a population of the transfected cells to create a stably transfected packaging and producer cell line.
  • the stably transfected producer cell line is capable of producing replication-deficient viral particles in association with the transgene.
  • the transfection comprises electroporation, nucleofection, lipofection, or calcium phosphate transfection.
  • the method is helper virus-free.
  • an enzyme capable of performing targeted genomic integration causes the transgene to be inserted in a certain genomic locus and/or site (e.g., at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS)) of a nucleic acid molecule.
  • the enzyme capable of performing targeted genomic integration is a recombinase.
  • the enzyme has one or more mutations which confer hyperactivity.
  • the recombinase is an integrase.
  • the recombinase is an integrase or a mobile element enzyme.
  • the integrase is a mobile element enzyme.
  • the mobile element enzyme is an engineered mammalian mobile element enzyme.
  • the mobile element enzyme is a mammal-derived RNA mobile element enzyme (e.g., a helper RNA mobile element enzyme).
  • the mobile element enzyme is a mammal-derived DNA mobile element enzyme.
  • the mobile element enzyme is a chimeric mobile element enzyme.
  • the enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
  • enzyme has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int-).
  • the mobile element enzyme comprises: (a) a gene-editing system, and (b) a mobile element enzyme that is capable of inserting the donor DNA comprising a transgene at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).
  • the transgene encodes a complete polypeptide.
  • the transgene is defective or substantially absent in a disease state.
  • the gene-editing system comprises a Cas9 enzyme guide RNA complex.
  • the Cas9 enzyme guide RNA complex comprises a nuclease-deficient (or inactive, or dead) dCas9 guide RNA complex, also referred to as dCas9 guide RNA complex.
  • the nuclease-deficient dCas9 guide RNA complex comprises a guide RNA selected from: GTTTAGCTCACCCGTGAGCC (SEQ ID NO: 91), CCCAATATTATTGTTCTCTG (SEQ ID NO: 92), GGGGTGGGATAGGGGATACG (SEQ ID NO: 93), GGATCCCCCTCTACATTTAA (SEQ ID NO: 94), GTGATCTTGTACAAATCATT (SEQ ID NO: 95), CTACACAGAATCTGTTAGAA (SEQ ID NO: 96), TAAGCTAGAGAATAGATCTC (SEQ ID NO: 97), and TCAATACACTTAATGATTTA (SEQ ID NO: 98), or a variant thereof.
  • the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme is an engineered version, including but not limited to hyperactive forms, of an enzyme (e.g., without limitation, a mobile element enzyme) derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • an enzyme e.g., without limitation, a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis
  • the mobile element enzyme is from one or more of the Sleeping beauty, Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1/mariner, or Tc3 donor DNA system, or biologically active fragments variants thereof, inclusive of hyperactive variants.
  • the enzyme is a mobile element enzyme such as a Myotis lucifugus mobile element enzyme (MLT mobile element enzyme), also referred to herein as an MLT mobile element enzyme.
  • MLT mobile element enzyme is a wild-type MLT mobile element enzyme.
  • the MLT mobile element enzyme is a modified MLT mobile element enzyme, also referred to herein as a corrected MLT mobile element enzyme.
  • the MLT mobile element enzyme e.g., the wild-type MLT mobile element enzyme, the corrected MLT mobile element enzyme, or a variant thereof
  • has one or more mutations such as hyperactive mutations.
  • the MLT mobile element enzyme is a modified MLT mobile element enzyme.
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, and one or more mutations selected from L573X, E574X, and S2X, wherein X is any amino acid or no amino acid, optionally X is A, G, or a deletion.
  • the mutations are L573del E574del, and S2A (SEQ ID NO: 1).
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1 and S8P and C13R mutations (SEQ ID NO: 11).
  • the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to at least one of S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to S8P, C13R, and N125K mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof.
  • the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2 (which is codon- optimized) and an amino acid sequence SEQ ID NO: 1.
  • the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or a codon- optimized form thereof.
  • the MLT mobile element enzyme has an amino acid sequence SEQ ID NO: 1, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the MLT mobile element enzyme includes a hyperactive mutation selected from TABLE 14A and TABLE 14B, e.g., about 1, or about 2, or about 3, or about 4, or about 5 hyperactive mutations selected from TABLE 14A or TABLE 14B, or combinations thereof.
  • the MLT mobile element enzyme is a wild-type mobile element enzyme that has an amino acid sequence of SEQ ID NO: 10.
  • the MLT mobile element enzyme is a modified mobile element enzyme having the amino acid sequence of SEQ ID NO: 11.
  • at least one of the first, second, and third nucleic acids is in the form of a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • a nucleic acid encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme) and a nucleic acid encoding a donor DNA are in the form of the same LNP, optionally in a co-formulation.
  • a nucleic acid encoding an enzyme capable of performing targeted genomic integration e.g., without limitation, a mobile element enzyme
  • a nucleic acid encoding a donor DNA are in the same mixture with an LNP.
  • a method of producing an AAV bearing a gene of interest is provided, to produce the AAV bearing the gene of interest.
  • the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression.
  • the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with REP expression or substantially enhanced REP expression.
  • the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression and REP expression or substantially enhanced REP expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with REP expression or substantially enhanced VP1 expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression. In embodiments, a cell for gene therapy is provided that is generated by a method in accordance with embodiments of the present disclosure. In some embodiments, a pharmaceutical composition comprising the cell is provided.
  • a method for treating a disease or condition using gene therapy comprising administering to a subject in need thereof a transfected cell generated using a method in accordance with embodiments of the present disclosure.
  • the disease or condition comprises cancer.
  • the disease or condition comprises an infectious disease.
  • the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof.
  • FIG.1A is a non-limiting representation of an AAV production strategy in accordance with embodiments of the present disclosure, using a donor vector to create a HEK293 producer cell line or another cell line expressing the AdV E1a and E1b genes (e.g., engineered CHO-K1 or Vero cell line with E1a, E1b) that forms a replication deficient AAV particle containing a transgene of interest.
  • the single or dual donor comprising a transgene of interest is incorporated (e.g., by transfection such as electroporation) into a HEK293 cell line or another cell line expressing the E1a and E1b genes.
  • FIG.1B is a representation of an example of an inducible Rep/Cap and helper AAV donor plasmid construct (Kana r ), encoding E2A, E4ORF6, and VA RNA helper genes flanked by insulators and mobile element enzyme recognition ends, and used with a helper RNA or DNA to create an AAV producer cell line, in accordance with embodiments of the present disclosure.
  • FIG.1C is a representation of an example of a nucleic acid (plasmid) encoding a transgene (gene of interest (GOI)) included between AAV inverted terminal repeats (ITRs), in accordance with embodiments of the present disclosure.
  • FIGs.2A-E depict non-limiting representations of chimeric, monomer or head-to-tail dimer mobile element enzymes that are designed to target human GSHS using TALE and Cas9/guide RNA DNA binders.
  • TALEs include nuclear localization signals (NLS) and an activation domain (AD) to function as transcriptional activators.
  • the DNA binding domain has approximately 16.5 repeats of 33-34 amino acids with a residual variable di-residue (RVD) at position 12-13.
  • RVD residual variable di-residue
  • FIG.2B RVDs are shown that have specificity for one or several nucleotides. Only bases of the DNA leading strand are shown.
  • FIG.2C A chimeric mobile element enzyme construct comprising a TALE DNA-binding protein fused thereto by a linker that is greater than 23 amino acids in length (top) and a chimeric mobile element enzyme construct comprising dCas9 linked to one or more guide RNAs (bottom).
  • 2D is a non-limiting representation of a system in accordance with embodiments of the present disclosure comprising a nucleic acid (e.g., helper RNA) encoding an enzyme capable of performing targeted genomic integration and a nucleic acid encoding a mobile element enzyme (donor DNA).
  • the helper RNA is translated into a bioengineered enzyme (e.g., integrase, recombinase, or mobile element enzyme) that recognizes specific ends and seamlessly inserts the donor DNA into the human genome in a site-specific manner without a footprint.
  • Chimeric mobile element enzymes form dimers or tetramers at open chromatin to insert donor DNA at TTAA recognition sites near DNA binding regions targeted by dCas9/gRNA or TALEs.
  • FIG.2E is a non-limiting representation of a system in accordance with embodiments of the present disclosure comprising a nucleic acid (e.g., helper RNA) encoding an enzyme capable of performing targeted genomic integration and a nucleic acid encoding a mobile element enzyme (donor DNA).
  • a nucleic acid e.g., helper RNA
  • the helper RNA is translated into a bioengineered enzyme (e.g., integrase, recombinase, or mobile element enzyme) that recognizes specific ends and seamlessly inserts the donor DNA into the human genome in a site-specific manner without a footprint.
  • FIG.3B depicts a donor DNA construct template with the transgene VLDLR.
  • FIG.3C depicts a chimeric mobile element enzyme construct template with a TALE DNA binder. Other TALEs and mobile element enzymes can be substituted.
  • FIG.3D depicts a chimeric mobile element enzyme construct template with a dCas9/gRNA DNA binder.
  • FIG.3E depicts a system comprising donor DNA (panel A) and helper RNA (panel B): panel (A)
  • the donor DNA can be any gene of interest (GOI) including a gene that replaces, inactivates, or provides suicide or helper functions.
  • the GOI can be driven by a predetermined promoter and flanked by insulators to prevent gene silencing.
  • the internal terminal repeats (ITRs) can be specific for a mammal- derived mobile element enzyme.
  • the helper RNA is 5’-m7G capped (cap 0, or cap1, or cap 2) with flanking globin 5’- and 3’-UTRs, an N-terminus nuclear localization signal (SV40 or nucleoplasmin), a 34 polyalanine tail region, and pseudouridine modification.
  • the mobile element enzyme e.g., hyperactive mobile element enzyme
  • FIG.4 depicts a non-limiting representation of a conventional rAAV production system.
  • Production in adenovirus complementation systems is usually performed as plasmid transfection processes, where AAV Rep/Cap genes, the ITR-flanked gene of interest (GOI), as well as Ad-helper genes are provided as three separate plasmids, respectively, to a E1a/E1b containing HEK293 cell line or other engineered cell line that contains E1a/E1b (e.g., engineered CHO- K1 or Vero cells).
  • AAV Rep/Cap genes the ITR-flanked gene of interest (GOI)
  • Ad-helper genes are provided as three separate plasmids, respectively, to a E1a/E1b containing HEK293 cell line or other engineered cell line that contains E1a/E1b (e.g., engineered CHO- K1 or Vero cells).
  • FIG.5 depicts a non-limiting schematic of an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promote with AAV2_REP52/40 ORFs and CAPs (VP1/2/3) under their native P19 or P40 promoter, respectively, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO- containing SV40 promoter, 3) VA RNAs under the control of HSV-2 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR engineered mobile element enzyme
  • FIG.6 depicts a non-limiting schematic of an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter and AAV2_REP52/40 ORFs under the native P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 4) VA RNAs under the control of HSV-2 ICP4 promoter, and 5) hygro-B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter and AAV2_REP52/40 ORFs under the native P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promote
  • FIG.7 depicts a non-limiting schematic of an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter and AAV2_REP52/40 ORFs under the native P19 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-2 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • FIG.8 depicts a non-limiting schematic of a construct encoding AAV2 CAP under the control of the tetO-containing hCMV-derived enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and modified AAV2 P40 intron_1 (a at position 276 substituted for C) and WPRE/modified ICP27 poly A, followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with ITRs for Sleeping Beauty (SB) transposase.
  • SB Sleeping Beauty
  • FIG.9 depicts a non-limiting schematic of a construct encoding AAV2 CAP under the control of the tetO-containing hCMV enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and MODIFIED AAV2 P40 intron_2 and WPRE/modified ICP27 poly A, followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs.
  • FIG.10A-10B depicts GFP images 120 hours post-transduction of AAVH clone 17+ (FIG.10A) and ddPCR analyses for quantification of the rAAV2-EGFP genome copy number per nanogram (ng) of total genomic DNA (FIG.10B).
  • FIG.11A – 11B depicts GFP images 120 hours post-transduction of AAVH clone 13 + (FIG.10A) and ddPCR analyses for quantification of the rAAV2-EGFP genome copy number per nanogram (ng) of total genomic DNA (FIG.10B).
  • FIG.12A – 12B depicts expression of functional AAV2 Rep proteins in AAV2-HB (Plasmid shown in FIG.6) clones as determined by western blot and ddPCR.
  • FIG.12A depicts a western blot analysis illustrating cells from selected AAV2- H B cell clones HB-36, HB-42, HB-118, and HB-120 were treated with10ng/ml doxycycline for 72 hours followed by extraction with RIPA buffer in the presence of proteinase inhibitor.
  • FIG.12B shows the results of a ddPCR analysis for AAV Genome Amplification / Rep function Assay.
  • FIG.13A – 13B depicts expression of functional AAV2 Rep proteins in AAV-CDH (Plasmid shown in FIG.7) clones as determined by western blot (FIG.13A) and ddPCR (FIG.13B).
  • FIG.14A – 14B depicts establishment of AAV2 full producer stable cell pools, CDH-24/Cap-SB, CDH-48/Cap-SB, and CDH-129/Cap-SB, with sleeping beauty transposase-mediated integration of AAV2 CAPs-expressing cassettes (Plasmid shown in FIG.7) into AAV-CDH Clone 24, 48, 129 serotype adaptable (Cap minus or Cap-) stable cell lines.
  • FIG.14A shows ddPCR analyses of SB-mediated AAV2 Cap transposon integration in each Zeocin-selected stable pools.
  • FIG.14B shows western blot analysis of SB-mediated AAV2 Cap transposon integration in each Zeocin-selected stable pools.
  • FIG.15A – 15B depicts expression of functional AAV2 Rep proteins in CDH-24/Cap-SB, CDH-48/Cap-SB, and CDH- 129/Cap-SB pools as determined by western blot and ddPCR.
  • FIG.15A shows ddPCR results of rAAV Rep proteins being expressed and
  • FIG.15B shows expressed AAV2 Rep proteins are functionally active in supporting AAV genome amplification.
  • FIG.16 depicts expression of AAV2 Cap and Rep in CDH-48/Cap-SB stable pool increased with doxycycline dosages and over time.
  • Cells from CDH-48/Cap-SB stable pools were incubated either in the presence of 0, 5, 10, 25, 50, or 100ng/ml doxycycline and harvest at 72 hours post induction; or incubated with 50ng/ml doxycycline and harvested at 24, 48, 72, or 96 hours post induction (FIG.16).
  • Cell extraction with RIPA buffer containing proteinase inhibitors, western blot, and ddPCR analyses of the CDH-48/Cap-SB (AAV2-capsid minus stable lines Clone CDH-48) samples were caried out as described previously.
  • FIG.17A – 17B depicts production of competent rAAV2 particles from stably engineered CDH-48/Cap-SB cell pools in response to doxycycline.
  • FIG.17A show a graph depicting rAAV2 physical titers from CDH-48/CAP-SB stable pool harvests.
  • FIG.17B shows a graph depicting transduction of CDH-48 cells (AAV2-CAPSID MINUS STABLE LINES CLONE CDH-48) with unconcentrated rAAV supernatents.
  • FIG.18A – 18B depicts characterization of Zeocin-resistant single clones selected from the CDH-48/Cap-SB stable pool.
  • FIG.18A depicts ddPCR analysis for SB-mediated AAV2-Cap cassette integration.
  • FIG.18B depicts western blot analysis with antibodies specific for AAV2 CAP.
  • FIG.19A – 19B depicts characterization of Zeocin-resistant single clones selected from the CDH-48/Cap-SB stable pool.
  • FIG.19A depicts western blot analysis with antibodies specific for AAV2 REP.
  • FIG.19B shows ddPCR assays of AAV genome amplification following transduction with low VGC/cell of AAV2-EGFP viral particles.
  • the present invention is based, in part, on the discovery that a mobile element enzyme-mediated site-specific genomic integration can be used to produce a stable AAV-based cell line.
  • the cell line is produced by insertion of inducible viral replication (rep) and Capsid (cap) genes and helper genes (E4ORF6, E2A, and VA) in specific genomic locations of a cell such as, e.g., a HEK293 (E1+) cell or a genetically engineered CHO-K1 or Vero cell line.
  • An advantage of the approach in accordance with embodiments of the present disclosure is that it does not require DNA homology for recombination, and it is independent of the size of the donor or recipient DNA molecules.
  • the described method is performed in a brief enzyme-catalyzed reaction using an enzyme (e.g., without limitation, a mobile element enzyme) delivered as either DNA or RNA.
  • an enzyme e.g., without limitation, a mobile element enzyme
  • this protocol allows for the production of stable expression human cell pools in about 4 weeks or in less than 4 weeks.
  • the present application provides methods of making viral particle packaging and producer cell lines.
  • the methods comprise transfecting a cell with two or more nucleic acids.
  • the nucleic acids encode one or more of: (a) an enzyme capable of performing targeted genomic integration, (b) an inducible viral replication (Rep) gene, (c) an inducible viral Capsid (cap) gene, (d) one or more adenoviral auxiliary genes (e.g., selected from one or more of E1A, E1B, E4, E2A, and VA of an AdV), (e) an insulator (e.g., selected from HS4, D4Z4), (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV inverted terminal repeats (ITRs).
  • an enzyme capable of performing targeted genomic integration e.g., an enzyme capable of performing targeted genomic integration
  • Rep inducible viral replication
  • Cap inducible viral Capsid
  • adenoviral auxiliary genes e.g., selected from one or more of E1A, E1B, E4, E2A, and VA of an AdV
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the resulting transfected cell expresses the transgene in association with the viral particle.
  • the cell is transfected with two nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV ITRs.
  • the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, (f) one or more terminal ends recognized by the enzyme, and (g) a transgene flanked by AAV ITRs.
  • the cell is transfected with three nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, and (f) one or more terminal ends recognized by the enzyme; and the third nucleic acid encodes (g) a transgene flanked by AAV ITRs.
  • the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration
  • the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, and (f) one or more terminal ends recognized by the enzyme
  • the third nucleic acid encodes (g) a transgene flanked by AAV ITRs.
  • the cell is transfected with four nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (d) one or more adenoviral auxiliary genes, (e) an insulator, and (f) one or more terminal ends recognized by the enzyme; the third nucleic acid encodes (c) an inducible viral cap gene; and the fourth nucleic acid encodes (g) a transgene flanked by AAV ITRs.
  • the present application provides methods of transfecting an E1A, E1B+ cell with two or more nucleic acids.
  • the nucleic acids encode one or more of: (a) an enzyme capable of performing targeted genomic integration, (b) an inducible viral replication (Rep) gene, (c) an inducible viral Capsid (cap) gene, (d) an insulator (e.g., selected from HS4, D4Z4), (e) one or more terminal ends recognized by the enzyme, and (f) a transgene flanked by AAV inverted terminal repeats (ITRs).
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the resulting transfected cell expresses the transgene in association with the viral particle.
  • the E1A, E1B+ cell is transfected with two nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration, and the second nucleic acid encodes an inducible viral Rep gene, (c) an inducible viral cap gene, (d) an insulator, (e) one or more terminal ends recognized by the enzyme, and (f) a transgene flanked by AAV ITRs.
  • the cell is transfected with three nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (c) an inducible viral cap gene, (d) an insulator, and (e) one or more terminal ends recognized by the enzyme; and the third nucleic acid encodes (f) a transgene flanked by AAV ITRs.
  • the cell is transfected with four nucleic acids, and the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration; the second nucleic acid encodes (b) an inducible viral Rep gene, (d) an insulator, and (e) one or more terminal ends recognized by the enzyme; the third nucleic acid encodes (c) an inducible viral cap gene; and the fourth nucleic acid encodes (f) a transgene flanked by AAV ITRs.
  • the first nucleic acid encodes (a) an enzyme capable of performing targeted genomic integration
  • the second nucleic acid encodes (b) an inducible viral Rep gene, (d) an insulator, and (e) one or more terminal ends recognized by the enzyme
  • the third nucleic acid encodes (c) an inducible viral cap gene
  • the fourth nucleic acid encodes (f) a transgene flanked by AAV ITRs.
  • the present application provides a method of making a viral particle packaging and producer cell line, the method comprising transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, a second nucleic acid encoding a donor DNA comprising inducible viral replication (Rep) and Capsid (cap) genes, adenoviral auxiliary genes selected from E1A and E1B, and insulators (optionally selected from HS4, D4Z4), and comprising terminal ends recognized by the enzyme, and a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs), to thereby result in a transfected cell that expresses the transgene in association with the viral particle.
  • ITRs inverted terminal repeats
  • a method of making a viral particle packaging and producer cell line comprises: transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration; a second nucleic acid encoding a donor DNA comprising inducible viral replication (rep) and Capsid (cap) genes, adenoviral auxiliary genes selected from E2A, E4ORF6, and VA, and insulators (optionally selected from HS4, D4Z4), and comprising terminal ends recognized by the enzyme, wherein the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof; and a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs), to thereby result in
  • a method of making a viral particle packaging and producer cell line comprises: transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and a second nucleic acid encoding viral packaging genes, viral helper genes, insulators (optionally selected from HS4 and D4Z4), and a transgene, and comprising terminal ends recognized by the enzyme to thereby result in a transfected cell that expresses the transgene in association with a viral particle.
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO- containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • a mammal-derived, helper RNA mobile element enzyme and donor DNA system is used to produce purified recombinant adeno-associated virus (rAAV) vector stocks.
  • rAAV stocks comprising an ITR-flanked therapeutic transgene or a gene of interest (GOI), are generated by a one plasmid or two-plasmid system, which creates a single producer cell line or separate packaging and producer cell lines.
  • the assembly of rAAV vectors typically requires (1) a recombinant vector genome comprising a gene of interest (GOI) and the regulation elements for the GOI expression in target cells (e.g., a promoter, poly A, introns, etc.) flanked by AAV ITRs, (2) the AAV rep and cap genes provided in trans, and (3) Helper functions from adeno-, herpes-, human papilloma- or vaccinia viruses, for replication and rescue of the recombinant genome.
  • GOI gene of interest
  • target cells e.g., a promoter, poly A, introns, etc.
  • helper functions from adeno-, herpes-, human papilloma- or vaccinia viruses for replication and rescue of the recombinant genome.
  • a conventional method for production of a vector is co-transfection of a vector plasmid containing the vector genome and a helper plasmid encoding the rep and cap genes into E1A-transformed human embryonic kidney cells (HEK293 cells) infected with Ad (see FIG.4).
  • HEK293 cells E1A-transformed human embryonic kidney cells
  • Ad Ad
  • AAV vector packaging strategies that rely on overexpression of the viral gene products face challenges such as issues associated with the cellular toxicity of Rep proteins, and the requirement that rep and cap expression levels be tightly regulated for cell viability hence maximal virion production.
  • An inducible promoter e.g., tetracycline (Tet), cumate, coumermycin/novobiocin, etc.
  • HEK293 cells are simultaneously transfected by a vector plasmid (containing the GOI) and one or two helper plasmids.
  • the helper plasmid(s) allow the expression of the four Rep proteins, the three AAV structural proteins VP1, VP2, and VP3, the AAP, and the adenoviral auxiliary functions E2A, E4ORF6, and VA RNA.
  • adenoviral E1A/E1B co-factors necessary for rAAV replication are expressed in HEK293 producer cells. See Qiao et al., J Virol 2002;76:1904-13; Yuan et al., Hum Gene Ther 2011;22:613-24; Lock et al., Hum Gene Ther 2010;21:1259- 71.
  • Rep/cap genes and adenoviral helper sequences are either cloned on two separate plasmids or combined on one plasmid, hence evolving from a triple plasmid system to transfection with only two plasmids.
  • the triple plasmid protocol provides versatility with a cap gene that can be switched from one serotype to another.
  • the plasmids are usually produced by conventional techniques in E. coli using bacterial origin and antibiotic-resistance gene or by minicircle (MC) technology.
  • Schnodt et al. Mol Ther Nucleic Acids 2016;5:e355.
  • transient transfection in adherent HEK293 cells has been used for large-scale manufacturing of rAAV vectors, it typically requires multiple production batches to fulfill the needs of clinical trials, resulting in lengthy and costly production campaigns.
  • HEK293 cells have been adapted to suspension conditions to be economically viable in the long term.
  • Grieger et al. Mol Ther 2016;24:287-97.
  • a method of making a viral particle packaging and producer cell line comprises transfecting an E1A, E1B+ cell with (1) a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, (2) a second nucleic acid encoding a donor DNA comprising inducible viral replication (rep) and Capsid (cap) genes, adenoviral auxiliary genes selected from E2A, E4ORF6, and VA, and insulators (optionally selected from HS4 and D4Z4), and comprising terminal ends recognized by the enzyme, wherein the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO-containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof, and (3) a third nucleic acid encoding a donor DNA comprising a transgene flank
  • a method of making a viral particle packaging and producer cell line comprises transfecting an E1A, E1B+ cell with (1) a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and (2) a second nucleic acid encoding viral packaging genes, viral helper genes, insulators (optionally selected from HS4 and D4Z4), and a transgene, and comprising terminal ends recognized by the enzyme, to thereby result in a transfected cell that expresses the transgene in association with a viral particle.
  • FIG.1A illustrates a non-limiting example of a method for generating AAV, using a single or dual donor DNA to create a producer cell line from a E1+ cell line (e.g., without limitation, an HEK293 cell line) that forms a replication deficient (rep-) AAV particle containing a transgene of interest.
  • a E1+ cell line e.g., without limitation, an HEK293 cell line
  • rep- replication deficient
  • a donor DNA construct encodes inducible Rep and Cap (“Capsid”) proteins (VP1, VP2, and VP3) and helper genes (E4, E2A, and VA) having end sequences recognized by an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme), as well as a transgene (or gene) of interest flanked by AAV ITRs.
  • the donor DNA construct is a dual donor DNA comprising a nucleic acid encoding the inducible Rep and Cap proteins and helper genes that is separate from a nucleic acid encoding transgene.
  • the donor DNA construct is a single donor DNA comprising a nucleic acid encoding the inducible Rep and Cap proteins, helper genes, and a transgene.
  • FIG.1B shows an example of an inducible Rep/Cap and helper AAV donor plasmid construct (Kana r ), encoding helper E2A, E4ORF6 and VA genes flanked by insulators and mobile element enzyme recognition ends, and used with a helper RNA or DNA (e.g., a mobile element enzyme) to create an AAV producer cell line.
  • FIG.1C shows an example of a plasmid encoding a transgene included between AAV ITRs.
  • the plasmids shown in FIGs.1B and 1C can be combined or can be used separately for transfection into an E1+ cell line to produce an AAV particle comprising the transgene (or gene of interest (GOI)).
  • the method depicted in FIG.1A is performed using a system in accordance with embodiments of the present disclosure shown in FIG.2D.
  • the system of FIG.2D is an integrative, non-viral donor DNA system for site- specific, stable genomic integration.
  • the system allows producing any recombinant AAV serotype by the insertion of inducible AAV rep/cap genes and helper genes (E4, E2A, and VA) in specific genomic locations in a cell, such as, e.g., HEK293 (E1+) cells, a genetically engineered CHO-K1 cell line, or in other cells.
  • the system of FIG.2D comprises comprising a nucleic acid (e.g., helper RNA [which is different from helper genes]) encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme) and a nucleic acid encoding a transgene of interest (donor DNA).
  • the helper RNA is translated into a bioengineered enzyme (e.g., integrase, recombinase, or mobile element enzyme) that recognizes specific ends and seamlessly inserts the transgene of interest into the human genome in a site-specific manner without a footprint.
  • a bioengineered enzyme e.g., integrase, recombinase, or mobile element enzyme
  • the present disclosure provides, in embodiments, methods for producing AAV packaging cell lines capable of regulated gene amplification.
  • the methods of the present disclosure employ tetracycline repressor-controlled (tetR) tetO containing promoters (Yao F et al., Hum Gene Ther., 9:1939-1950, 1998) or reverse tetracycline-controlled transactivator (rtTA)-controlled gene expression and SV40 origin replication to regulate the expression of the AAV rep and cap genes, and the AdV5 E2A and E4ORF6.
  • tetR tetracycline repressor-controlled
  • rtTA reverse tetracycline-controlled transactivator
  • binding of doxycycline to t-REx or rtTA will initiate transcription from the tetO-containing promoters or SV40 T-antigen gene, leading to expression of the AAV rep and cap genes, and AdV5, E2A, and E4ORF6 genes (FIG. 1B).
  • the construct shown in FIG.1B is permanently integrated into a E1a/E1b containing cell line (e.g., without limitations, an HEK293 cell line stably expressing tetR gene).
  • Transfection in media with an inducer (e.g., without limitation, doxycycline) of inducible rep and cap genes results in high-level production of Rep and Cap proteins and AAV virion assembly.
  • producer cells can be generated from these packaging lines by the addition of a vector construct comprising a transgene (or a GOI) flanked by AAV ITRs that can excise, replicate, and be packaged into virions once rep and cap are expressed.
  • the transgene construct is included in the same plasmid as the donor DNA, in a “single plasmid” protocol.
  • methods for an AAV packaging and producer cell line are provided.
  • the AAV rep and cap genes are under control of an inducible promoter (e.g., t-Rex or rtTA), such that the rep and cap genes, the AAV vector DNA sequences, and the essential helper genes are encoded on a single donor plasmid flanked by mobile element enzyme recognition sequences (FIG.1B).
  • a transgene is encoded separately from the donor plasmid (see, e.g., FIG.1C).
  • the donor plasmid comprises a transgene such that a one-plasmid (e.g., without limitation, a minicircle) is created and used to generate a transgene-specific cell line (e.g., an E1A, E1B+ cell such as, without limitation, a HEK293 cell line) that is able to produce a clinical grade product for human gene therapy.
  • a transgene-specific cell line e.g., an E1A, E1B+ cell such as, without limitation, a HEK293 cell line
  • methods for making a packaging and producer cell line with a dual donor/helper system are provided.
  • inducible Rep and Cap genes (sometimes collectively referred to as “Rep/Cap genes”), and a transgene of interest are encoded by respective separate nucleic acids.
  • the dual donor/helper system includes, without limitation, a nucleic acid encoding inducible Rep and Cap genes, a nucleic acid (e.g., a donor expression vector, such as a helper AAV donor plasmid) encoding a transgene of interest, and a nucleic acid encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme).
  • a method of making a viral particle packaging and producer cell line is provided.
  • the method comprises transfecting an E1A, E1B+ cell with: a first nucleic acid encoding an enzyme capable of performing targeted genomic integration; a second nucleic acid encoding a donor DNA comprising inducible viral replication (Rep) and Capsid (Cap) genes, adenoviral auxiliary genes selected from E2A, E4ORF6, and VA, and insulators (e.g., without limitation, HS4 or D4Z4), and comprising terminal ends recognized by the enzyme; and a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs), to thereby result in a transfected cell that expresses the transgene in association with the viral particle.
  • ITRs inverted terminal repeats
  • inducible Rep/Cap genes and a nucleic acid e.g., a donor expression vector, such as a helper AAV donor plasmid
  • a nucleic acid e.g., a donor expression vector, such as a helper AAV donor plasmid
  • the single donor/helper system includes, without limitation, a nucleic acid (e.g., a donor expression vector) encoding inducible Rep and Cap genes and a transgene of interest, and a nucleic acid encoding an enzyme capable of performing targeted genomic integration (e.g., without limitation, a mobile element enzyme).
  • a method of making a viral particle packaging and producer cell line comprises transfecting an E1A, E1B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration; and a second nucleic acid encoding viral packaging genes, viral helper genes, insulators (e.g., without limitation, HS4, D4Z4), and a transgene, and comprising terminal ends recognized by the enzyme, to thereby result in a transfected cell that expresses the transgene in association with a viral particle.
  • insulators e.g., without limitation, HS4, D4Z4
  • the E1A, E1B+ cell is, without limitation, HEK293, E1A, E1B+ engineered CHO-K1, or Sf9 cell line.
  • the viral helper genes comprise adenoviral auxiliary genes.
  • the adenoviral auxiliary genes are selected from one or more of E1A, E1B, E4, E2A, and VA of an AAV, optionally E1A and E1B.
  • the viral packaging genes comprise rep and cap genes of viral replication (Rep) and Capsid (Cap) proteins of an AAV.
  • the expression of the Rep and Cap proteins is controlled by an inducible promoter, optionally an antibiotic-dependent promoter.
  • the antibiotic-dependent promoter is tetracycline- or a variant thereof dependent promoter and a tetracycline repressor-controlled (tetR) or tetracycline-controlled transactivator (rtTA) system is used.
  • the antibiotic-dependent promoter is a coumermycin/novobiocin promoter, or a variant thereof.
  • the inducible viral replication (Rep) and Capsid (cap) genes are controlled by at least one of a tetO- containing P5 promoter, P19 promoter, SV40 promoter, P40 promoter, or a functional variant or fragment thereof.
  • the tetO-containing AAV2 P40 promoter comprises a nucleotide sequence of SEQ ID NO: 816, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the P40 intron is an AAV2 P40 intron.
  • the AAV2 P40 intron comprises a C276A substitution, or a substitution at position corresponding thereto relative to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises one or more mutated translation start sites (ATGs), optionally wherein the translation start sites are mutated to one of CTG, ACG, and TTG.
  • the AAV2 P40 intron comprises substitutions at one or more positions A13, A32, T42, A61, A71, A89, A203, A246, A258, and T282, or one or more positions corresponding thereto, relative to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises substitutions at one or more positions A13C, A32C, T42C, A61C, A71T, A89C, A203T, A246C, A258C, and T282C corresponding to SEQ ID NO: 818.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 818, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 819, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 817, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified TATA box element has the nucleotide sequence of TATATAA.
  • the tetO- containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV2 P40 intron comprises the nucleotide sequence of SEQ ID NO: 823, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the tetO-containing P40 promoter is a tetO-containing AAV2 P40 promoter.
  • the AAV2 P40 promoter comprises the nucleotide sequence of SEQ ID NO: 820, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified P40 intron is a AAV9 P40 intron.
  • the modified AAV9 P40 intron comprises the nucleotide sequence of SEQ ID NO: 821, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the modified TATA box element has the nucleotide sequence of TATATAA.
  • the tetO-containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV9 P40 intron comprises the nucleotide sequence of SEQ ID NO: 822, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the tetO-containing AAV2 P40 promoter or a functional variant or fragment thereof, comprising a modified TATA box element and a modified AAV9 P40 intron comprises a nucleotide sequence of SEQ ID NO: 824, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the P40 promoter further comprises one or more enhancer elements.
  • the enhancer element comprises one or more cis-acting elements, optionally selected from an Sp1 binding site, GC rich sequence, GCGGAAC motif, TAATGARAT element, AP1 binding site, and CCAAT box element.
  • the enhancer element comprises about 1 to about 5 Sp1 binding sites, optionally about 1, or about 2, or about 3, or about 4, or about 5 Sp1 binding sites.
  • the enhancer element comprises about 1 or about 2 GC rich sequences.
  • the enhancer element is derived from an hCMV Enhancer Element-3.
  • the hCMV Enhancer Element-3 comprises the nucleotide sequence of SEQ ID NO: 830 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the viral particle is an AAV of a suitable serotype.
  • the AAV is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9.
  • the AAV serotype is a natural serotype or a synthetically engineered serotype.
  • the AAV is AAV2.
  • the AAV is AAV9.
  • the viral particle is an AAV of any of the AAV serotypes. Today, 12 AAV serotypes are known, and more than 100 variants have been identified.
  • any one or more of the first, second, and third nucleic acids is encoded by a single nucleic acid.
  • the second nucleic acid and the third nucleic acid are included in a single expression vector.
  • the first and second nucleic acids are included in a single expression vector, and the third nucleic acid is included in an expression vector that is different from the expression vector including the first and second nucleic acids.
  • the single expression vector comprises a plasmid.
  • the plasmid comprises an AAV2 CAP under the control of the tetO-containing hCMV-derived enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and modified AAV2 P40 intron_1 (a at position 276 substituted for C) and WPRE/modified ICP27 poly A followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 825 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an AAV2 CAP under the control of the tetO-containing hCMV enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and MODIFIED AAV2 P40 intron_2 and WPRE/modified ICP27 poly A followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 826 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2)Ad5 E2A_IRES_Ad5 E4 OR F6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-2 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2)Ad5 E2A_IRES_Ad5 E4 OR F6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-2 ICP4 promoter, and 4) hygro-
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 841 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, and CAPs under the native P40 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, and CAPs under the native P40 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 842, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 4) VA RNAs under the control of HSV-1 ICP4 promoter, and 5) hygro- B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 843, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • any one of the first, second, and third nucleic acids is RNA, optionally mRNA, optionally synthetic mRNA. In embodiments, any one of the first, second, and third nucleic acids is DNA, optionally plasmid DNA. In embodiments, any one of the first, second, and third nucleic acids is an expression vector, wherein the expression vector is optionally a plasmid.
  • the transfected cell generates using the method of making a viral particle packaging and producer cell line in accordance with embodiments of the present disclosure comprises a donor DNA comprising the transgene flanked by AAV inverted terminal repeats (ITRs).
  • the method further comprises culturing the transfected cell in a medium that expands a population of the transfected cells to create a stably transfected packaging and producer cell line.
  • the stably transfected producer cell line is capable of producing replication-deficient viral particles in association with the transgene.
  • the transfection comprises electroporation, nucleofection, lipofection, or calcium phosphate transfection.
  • the method is helper virus-free.
  • the cell is human embryonic kidney (HEK293), Chinese hamster ovary (CHO) E1A, E1B+ engineered CHO-K1, or Spodoptera frugiperda (Sf9) cell line, baby hamster kidney (BHK), vero cell.
  • the viral helper genes comprise adenoviral auxiliary genes.
  • the adenoviral auxiliary genes are selected from one or more of E1A, E1B, E4, E2A, and VA of an AAV, optionally E1A and E1B.
  • the viral packaging genes comprise rep and cap genes of viral replication (Rep) and Capsid (Cap) proteins of an AAV.
  • the expression of the Rep and Cap proteins is controlled by an inducible promoter.
  • the inducible promoter is an antibiotic-dependent promoter, optionally a tetracycline-dependent promoter or a variant thereof, or a cumate or coumermycin/novobiocin promoter or a variant thereof.
  • the viral particle is an AAV, and optionally wherein the AAV is selected from AAV1, AAV5, AAV2, AAV6, AAV7, AAV8, and AAV9.
  • any one or more of the first, second, and third nucleic acids is encoded by a single nucleic acid.
  • the second nucleic acid and the third nucleic acid are included in a single expression vector.
  • the first and second nucleic acids are included in a single expression vector, and the third nucleic acid is included in an expression vector that is different from the expression vector including the first and second nucleic acids.
  • the expression vector is or comprises a plasmid.
  • the plasmid comprises an AAV2 CAP under the control of the tetO-containing hCMV-derived enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and modified AAV2 P40 intron_1 (a at position 276 substituted for C) and WPRE/modified ICP27 poly A followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 825 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an AAV2 CAP under the control of the tetO-containing hCMV enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and MODIFIED AAV2 P40 intron_2 and WPRE/modified ICP27 poly A followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs.
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 826 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2)Ad5 E2A_IRES_Ad5 E4 OR F6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, 2)Ad5 E2A_IRES_Ad5 E4 OR F6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 841 or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, and CAPs under the native P40 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter, REP 52/40 under the control of P19 promoter, and CAPs under the native P40 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 842, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the plasmid comprises an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter and REP 52/40 under the control of P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 4) VA RNAs under the control of HSV-1 ICP4 promoter, and 5) hygro- B resistant gene under the control of TK promoter.
  • ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter and REP 52/40 under the control of P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_
  • the plasmid comprises the nucleotide sequence of SEQ ID NO: 843, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • any one of the first, second, and third nucleic acids is or comprises RNA, optionally mRNA, optionally synthetic mRNA or modified mRNA. In embodiments, any one of the first, second, and third nucleic acids is DNA, optionally plasmid DNA. In embodiments, any one of the first, second, and third nucleic acids is an expression vector, wherein the expression vector is optionally a plasmid.
  • the transgene is flanked by AAV inverted terminal repeats (ITRs). In embodiments, the transgene is flanked by Sleeping beauty ITRs.
  • SEQ ID NO: 831 Sleeping Beauty LE ITR Sequence (IR/DR(L) Lmut44) (231 bp including TATA): 5’- tatacagttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactactccacaaatttcttgttaacaaacaatagttttggcaagtcagttag gacatctactttgtgcatgacacaagtcatttttccaacaattgtttacagacagattatttcacttataattcactgtatcacaattccagtgggtcagaagtttacatacact aa– 3’
  • the transgene or transposon has a size of about 200,000 base pairs or less. In embodiments, the transgene or transposon has a size of at least 200,000 base pairs. In embodiments, the transgene or transposon has a size of about 200,000 base pairs. In embodiments, the transgene or transposon has a size of about 150,000 base pairs or less. In embodiments, the transgene or transposon has a size of at least 150,000 base pairs. In embodiments, the transgene or transposon has a size of about 150,000 base pairs. In embodiments, the transgene or transposon has a size of about 100,000 base pairs or less. In embodiments, the transgene or transposon has a size of at least 100,000 base pairs.
  • the transgene or transposon has a size of about 100,000 base pairs. In embodiments, the transgene or transposon has a size of about 75,000 base pairs or less. In embodiments, the transgene or transposon has a size of at least 75,000 base pairs. In embodiments, the transgene or transposon has a size of about 75,000 base pairs. In embodiments, the transgene or transposon has a size of about 50,000 base pairs or less. In embodiments, the transgene or transposon has a size of at least 50,000 base pairs. In embodiments, the transgene or transposon has a size of about 50,000 base pairs.
  • the method of the present disclosure further comprises culturing the transfected cell in a medium that expands a population of the transfected cells to create a stably transfected packaging and producer cell line.
  • the stably transfected producer cell line is capable of producing replication-deficient viral particles in association with the transgene.
  • the transfection comprises electroporation, nucleofection, lipofection, or calcium phosphate transfection.
  • the method is helper virus-free.
  • the enzyme capable of performing targeted genomic integration is a recombinase.
  • the recombinase is an integrase or a mobile element enzyme.
  • the enzyme is a mobile element enzyme.
  • the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme is an engineered version, including but not limited to hyperactive forms, of an enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • an enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molos
  • the mobile element enzyme is from one or more of the Sleeping beauty, Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1/mariner, or Tc3 donor DNA system, or biologically active fragments variants thereof, inclusive of hyperactive variants.
  • the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • SEQ ID NO: 1 MLT mobile element enzyme protein (amino acid sequence of a variant of the hyperactive mobile element enzyme with S at position 8 and C at position 13 (572 amino acids) MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEWSHV DNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDITPQEMKK FLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKVRPVLDYFVP KFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGEGKRLLETIQTVV SPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFIRKNDILL
  • the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A.
  • the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1.
  • the enzyme has one or more mutations which confer hyperactivity.
  • the enzyme has one or more amino acid substitutions selected from S8X 1 and/or C13X 2 , or positions corresponding thereto relative to SEQ ID NO: 1.
  • the enzyme has S8X 1 and/or C13X 2 substitutions, at positions corresponding thereto relative to SEQ ID NO: 1.
  • the enzyme has S8X 1 and C13X 2 substitutions, at positions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme has S8X 1 substitution, at position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme has C13X 2 substitution, at positions corresponding thereto relative to SEQ ID NO: 1. In embodiments, X 1 is selected from G, A, V, L, I, and P and X 2 is selected from K, R, and H. In embodiments, X 1 is P and X 2 is R. In embodiments, the enzyme of the present disclosure comprises an amino acid sequence of SEQ ID NO: 11.
  • the enzyme comprises at least one, at least five, at least seven, at least nine, or ten substitutions at positions corresponding to: 5, 8, 9, 10, 11, 14, 22, 36, 37, 54, 130, 239, 281, 282, 283, 284, 285, 294, 300, 310, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 345, 375, 416, 427, 475, 481, 491, 520, and/or 561 of SEQ ID NO: 11.
  • the enzyme comprises one, two, three, four, five, six, seven, eight, nine, or ten substitutions at positions corresponding to: 5, 8, 9, 10, 11, 14, 22, 36, 37, 54, 130, 239, 281, 282, 283, 284, 285, 294, 300, 310, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 345, 375, 416, 427, 475, 481, 491, 520, and/or 561 of SEQ ID NO: 11.
  • the enzyme comprises at least one, at least five, at least seven, at least nine, or ten substitutions selected from S5P, S8F, D9G, D10G, E11G, A14V, T22C, S36G, T37C, S54N, K130T, G239R, Y281A, C282A, G283A, E284A, G285A, T294A, T300A, N310A, G330A, T331A, I332A, R333A, K334A, N335A, R336A, G337A, I338A, P339A, I345V, T375G, D416A, R427H, D475G, M481V, P491Q, A520T, and A561T, wherein the positions are corresponding to positions of SEQ ID NO: 11.
  • the enzyme comprises one, two, three, four, five, six, seven, eight, nine, or ten substitutions selected from S5P, S8F, D9G, D10G, E11G, A14V, T22C, S36G, T37C, S54N, K130T, G239R, Y281A, C282A, G283A, E284A, G285A, T294A, T300A, N310A, G330A, T331A, I332A, R333A, K334A, N335A, R336A, G337A, I338A, P339A, I345V, T375G, D416A, R427H, D475G, M481V, P491Q, A520T, and A561T, wherein the positions are corresponding to positions of SEQ ID NO: 11.
  • the mobile element enzyme is an engineered mammalian mobile element enzyme. In embodiments, the mobile element enzyme is a mammal-derived, helper RNA mobile element enzyme. In embodiments, the mobile element enzyme is a mammal-derived, helper DNA mobile element enzyme. In embodiments, the enzyme is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the enzyme is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site.
  • TTAA SEQ ID NO: 440
  • the mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+), and the mobile element enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 430, or a nucleotide sequence encoding the same.
  • the mobile element enzyme has one or more mutations which confer hyperactivity.
  • the mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11 or a functional equivalent thereof.
  • the mobile element enzyme has the nucleotide sequence having at least about 90% identity to SEQ ID NO: 5 or a codon-optimized form thereof.
  • the mobile element enzyme has an amino acid sequence having I83P and/or V118R mutation relative to the amino acid sequence of SEQ ID NO: 6 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having S20P and/or A29R mutation relative to the amino acid sequence of SEQ ID NO: 7 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having A12P and/or I28R mutation and/or R152K mutation relative to the amino acid sequence of SEQ ID NO: 9 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having T4P and/or L13R mutation relative to the amino acid sequence of SEQ ID NO: 8 or a functional equivalent thereof.
  • the donor DNA is included in a vector comprising left and right end sequences recognized by the mobile element enzyme.
  • the end sequences are selected from MER, MER75A, MER75B, and MER85.
  • the end sequences are selected from nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 441, and SEQ ID NO: 22, or a nucleotide sequence having at least about 90% identity thereto.
  • SEQ ID NO: 12 Pteropus vampyrus Left End Sequence Sequence 381 bp.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 12, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 12 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 17, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 17 is positioned at the 3’ end of the donor DNA.
  • the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 13, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 13 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 18, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 18 is positioned at the 3’ end of the donor DNA.
  • the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 14, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 14 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 19, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 19 is positioned at the 3’ end of the donor DNA.
  • the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 15, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 15 is positioned at the 5’ end of the donor DNA.
  • end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 20, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 20 is positioned at the 3’ end of the donor DNA.
  • the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 16, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 16 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441 is positioned at the 3’ end of the donor DNA.
  • the mobile element enzyme is an engineered form of a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
  • the enzyme is in a monomeric or dimeric form.
  • the enzyme is in a multimeric form.
  • the method of the present disclosure provides an enzyme comprising (a) a targeting element, and (b) an enzyme that is capable of inserting the donor DNA comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS).
  • the donor DNA comprises a transgene encoding a complete polypeptide.
  • the donor DNA comprises a transgene which is defective or substantially absent in a disease state.
  • the enzyme has one or more mutations which confer hyperactivity.
  • the enzyme has gene cleavage (Exc) and/or gene integration activity (Int+).
  • the enzyme has gene cleavage (Exc) and/or a lack of gene integration activity (Int-).
  • the mobile element enzyme is a chimeric mobile element enzyme.
  • the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a TnsD.
  • TALE transcription activator-like effector
  • the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD).
  • TALE DBD comprises one or more repeat sequences.
  • the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids.
  • the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.
  • RVD recognizes one base pair in the nucleic acid molecule.
  • the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome.
  • the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.
  • AAVS1 adeno-associated virus site 1
  • CCR5 chemokine receptor 5
  • HIV-1 coreceptor HIV-1 coreceptor
  • Rosa26 locus human Rosa26 locus.
  • the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the targeting element comprises a Cas9 enzyme guide RNA complex.
  • the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.
  • the targeting element comprises a Cas12 enzyme guide RNA complex or wherein the targeting element comprises a nuclease- deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex.
  • the targeting element comprises: a gRNA of or comprising a sequence of TABLE 3A-3F, or a variant thereof; or a TALE DBD of or comprising a sequence of TABLE 4A-4F, or a variant thereof; or a ZNF of or comprising a sequence of TABLE 5A-5E, or a variant thereof.
  • the targeting element is or comprises a nucleic acid binding component of the gene-editing system.
  • the enzyme and the targeting element are connected.
  • the enzyme and the targeting element are fused to one another or linked via a linker to one another.
  • the linker is a flexible linker.
  • the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is from about 1 to about 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.
  • the donor DNA comprises a gene encoding a complete polypeptide. In embodiments, the donor DNA comprises a gene which is defective or substantially absent in a disease state. In embodiments, the donor DNA is flanked by one or more inverted terminal ends. In embodiments, at least one of the first nucleic acid and the second nucleic acid is in the form of a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the first nucleic acid encoding the enzyme and the second nucleic acid encoding the donor DNA are in the form of the same LNP, optionally in a co-formulation.
  • the LNP comprises one or more lipids selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero- 3-methoxypolyethyleneglycol – 2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol-3phosphocholine (DSPC) and/or comprising of one or
  • DOTAP
  • the enzyme is encoded by a recombinant or synthetic nucleic acid.
  • the nucleic acid is mRNA or a helper RNA.
  • the nucleic acid is RNA that has a 5’-m7G cap (cap0, cap1, or cap2) with pseudouridinesubstitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.
  • the enzyme is incorporated into a vector or a vector-like particle.
  • the vector is a non- viral vector.
  • the enzyme and the donor DNA are included in the same vector. In embodiments, the enzyme and the donor DNA are included in different vectors.
  • the enzyme and the donor DNA are included in a single pharmaceutical composition. In embodiments, the enzyme and the donor DNA are included in different pharmaceutical compositions. In embodiments, the enzyme and the donor DNA are co- administered. In embodiments, the enzyme and the donor DNA are administered separately.
  • the present disclosure provides a method of producing an AAV bearing a gene of interest, comprising employing a method of the present disclosure to produce the AAV bearing the gene of interest. In embodiments, the present disclosure provides a cell for gene therapy, generated by a method of the present disclosure. In embodiments, the present disclosure provides a method of delivering a cell therapy, comprising administering to a patient in need thereof the transfected cell generated by a method of the present disclosure.
  • the present disclosure provides a method of treating a disease or condition using a cell therapy, comprising administering to a patient in need thereof the transfected cell generated by a method of the present disclosure.
  • the present disclosure provides a method of treating a disease or condition using a biologic, e.g., antibody, therapy, comprising administering to a patient in need thereof the transfected cell generated by a method of the present disclosure.
  • a biologic e.g., antibody
  • the enzyme capable of targeted genomic integration is any type of an enzyme that cause a transgene to be inserted from one location (e.g., without limitation, donor DNA) to a specific site and/or locus in a subject’s genome.
  • the enzyme capable of targeted genomic integration is a recombinase.
  • the recombinase is an integrase.
  • the enzyme is a mobile element enzyme.
  • the recombinase is an integrase or a mobile element enzyme.
  • the mobile element enzyme is an engineered mammalian mobile element enzyme.
  • the mobile element enzyme is a mammal-derived, helper RNA mobile element enzyme.
  • Messenger RNA (mRNA) is an effective alternative to DNA as a source of a mobile element enzyme for targeting somatic cells and tissues, given that RNA is a safer alternative to DNA as a source of a mobile element enzyme for somatic gene therapy applications.
  • the mobile element enzyme is a mammal-derived, DNA mobile element enzyme. In embodiments, the mobile element enzyme is a chimeric mobile element enzyme.
  • the enzyme capable of targeted genomic integration is a mobile element enzyme
  • the mobile element enzyme comprises (a) a targeting element that is or comprises a gene-editing system, and (b) a mobile element enzyme that is capable of inserting the donor DNA (e.g., mobile element) comprising a transgene at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a GSHS, as described elsewhere herein.
  • the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme is an engineered version, including but not limited to hyperactive forms, of an enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • an enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molos
  • the enzyme is a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme is an engineered version, including but not limited to hyperactive forms, of a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuh
  • the mobile element enzyme is from one or more of the Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1/mariner, or Tc3 donor DNA system, or biologically active fragments variants thereof, inclusive of hyperactive mutants (e.g., without limitation selected from TABLE 1, or equivalents thereof).
  • the mobile element enzyme is from a MLT donor DNA system that is based on a cut-and-paste MLT element obtained from the little brown bat (Myotis lucifugus) or other bat mobile element enzymes, such as Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii, and Molossus molossus.
  • MLT donor DNA system that is based on a cut-and-paste MLT element obtained from the little brown bat (Myotis lucifugus) or other bat mobile element enzymes, such as Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii, and Molossus molossus.
  • hyperactive forms of a bat mobile element enzyme are used.
  • the MLT mobile element enzyme has been shown to be capable of transposition in bat, human, mammalian, and yeast cells.
  • the hyperactive forms of the MLT mobile element enzyme enhance the transposition process.
  • chimeric MLT mobile element enzymes are capable of site-specific excision without genomic integration.
  • the mobile element enzyme is a Myotis lucifugus mobile element enzyme (MLT), which is either the wild type, monomer, dimer, tetramer (or another multimer), hyperactive, an Int-mutant, or of any other form.
  • MMT Myotis lucifugus mobile element enzyme
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant having at least about 80%, at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, and one or more mutations selected from L573X, E574X, and S2X, wherein X is any amino acid or no amino acid, optionally X is A, G, or a deletion, optionally the mutations are L573del E574del, and S2A).
  • the MLT mobile element enzyme has the nucleotide sequence of SEQ ID NO: 2 (which is a codon-optimized form of MLT), or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • SEQ ID NO: 1 is: MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEWSHV DNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDITPQEMKK FLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKVRPVLDYFVP KFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGEGKRLLETIQTVV SPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFIRKNDILLQVWQSKKP VYLISSIHSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDRADQYLS
  • the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A.
  • the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1.
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1 or a variant having at least about 80%, at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and comprises an alanine at the position corresponding to position 2 of SEQ ID NO: 1.
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1 or a variant having at least about 80%, at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and comprises an alanine at the position corresponding to position 2 of SEQ ID NO: 1 and no additional amino acids at the C terminal end.
  • the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2 (which is codon- optimized) and an amino acid sequence SEQ ID NO: 1, respectively.
  • the MLT mobile element enzyme has a nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or a codon-optimized form thereof.
  • the MLT mobile element enzyme has an amino acid sequence SEQ ID NO: 1, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the mobile element enzyme can act on an MLT left terminal end, or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, wherein the nucleotide sequence of the MLT left terminal end (5’ to 3’) is as follows: ttaacacttggattgcgggaaacgagttaagtcggctcgcgtgaattgcgcgcgtactccgcgggagccgtcttaactcggttcatatag atttgcggtggagtgcgggaaacgtgtaaactcgggccgattgtaactgcgtattaccaaatatttgtt (SEQ ID NO: 21)
  • the mobile element enzyme can act on an MLT right terminal end, or a sequence having at least about 90%, or at least about
  • the donor DNA is or comprises a gene encoding a compete polypeptide. In embodiments, the donor DNA is or comprises a gene which is defective or substantially absent in a disease state.
  • the enzyme e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme
  • the enzyme has one or more mutations which confer hyperactivity.
  • the enzyme e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme
  • the enzyme e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme
  • the mobile element enzyme e.g., without limitation, MLT mobile element enzyme includes a hyperactive mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 hyperactive mutations or combinations thereof.
  • the mobile element enzyme can include any number of any of the hyperactive mutations, or equivalents thereof, described herein.
  • the MLT mobile element enzyme includes a hyperactive mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 hyperactive mutations, or combinations thereof.
  • the mobile element enzyme can include any number of any of the hyperactive mutations, or equivalents thereof, described herein.
  • the enzyme comprises one or more mutations corresponding to TABLE 1, which, without being bound by theory, provides hyperactive mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1, and nucleic acid sequence of SEQ ID NO: 2.
  • the MLT mobile element enzyme has one or more amino acid substitutions selected from S8X1, C13X2 and/or N125X3, or positions corresponding thereto, relative to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
  • the MLT mobile element enzyme has S8X1, C13X2 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
  • the MLT mobile element enzyme has S8X1 and C13X2 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
  • the MLT mobile element enzyme has S8X1 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
  • the MLT mobile element enzyme has C13X2 and N125X3 substitutions, at positions corresponding to SEQ ID NO: 1, wherein X1 is selected from G, A, V, L, I and P, X2 is selected from K, R, and H, and X3 is selected from K, R, and H, or wherein: X1 is P, X2 is R, and/or X3 is K.
  • the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant thereof, and S8P and C13R mutations (SEQ ID NO: 11).
  • the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to at least one of S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence having mutations at positions which correspond to S8P and C13R mutations relative to the amino acid of SEQ ID NO: 1 or a functional equivalent thereof. In embodiments, the MLT mobile element enzyme has an amino acid sequence of SEQ ID NO: 1, or a variant thereof, and S8P, C13R, and N125K mutations (SEQ ID NO: 10).
  • a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more hyperactive mutations selected from a substitution or deletion at one or more of positions S5, S8, D9, D10, E11, C13, A14, S36, S54, N125, K130, G239, T294, T300, I345, R427, D475, M481, P491, A520, and A561, or positions corresponding thereto.
  • a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more hyperactive mutations selected from S5P, S8P, S8P/C13R, D9G, D10G, E11G, C13R, A14V, S36G, S54N, N125K, K130T, G239S, T294A, T300A, I345V, R427H, D475G, M481V, P491Q, A520T, and A561T, or positions corresponding thereto.
  • the MLT mobile element enzyme comprises one or more of hyperactive mutants selected from S8X 1 , C13X 2 and/or N125X 3 (e.g., all of S8X 1 , C13X 2 and N125X 3 , S8X 1 and C13X 2 , S8X 1 and N125X 3 , and C13X 2 and N125X 3 ), where X 1 , X 2 , and X 3 is each independently any amino acid, or X 1 is a non-polar aliphatic amino acid, selected from G, A, V, L, I and P, X 2 is a positively charged amino acid selected from K, R, and H, and/or X 3 is a positively charged amino acid selected from K, R, and H.
  • S8X 1 , C13X 2 and/or N125X 3 e.g., all of S8X 1 , C13X 2 and N125X 3 , S8X 1 and C13X 2 , S8X 1 and N125X 3 , and
  • X 1 is P
  • X 2 is R
  • X 3 is K
  • the enzyme e.g., without limitation, a mobile element enzyme, e.g., without limitation, MLT mobile element enzyme
  • the enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
  • the enzyme e.g., without limitation, a mobile element enzyme
  • the MLT mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
  • the MLT mobile element enzyme has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int-).
  • the mobile element enzyme e.g., without limitation, MLT mobile element enzyme includes an integration reduced or deficient mutation, e.g., about 1, or about 2, or about 3, or about 4, or about 5 integration reduced or deficient mutations or combinations thereof.
  • the mobile element enzyme can include any number of any of the integration reduced or deficient mutations, or equivalents thereof, described herein.
  • the MLT mobile element enzyme includes a integration reduced or deficient mutations, e.g. about 1, or about 2, or about 3, or about 4, or about 5 integration reduced or deficient mutations, or combinations thereof.
  • the mobile element enzyme can include any number of any of the integration reduced or deficient mutations, or equivalents thereof, described herein.
  • the enzyme comprises one or more mutations corresponding to TABLE 2A, or positions corresponding thereto, which, without being bound by theory, provides integration reduced or deficient mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1.
  • TABLE 2A In embodiments, the enzyme comprises one or more mutations corresponding to TABLE 2B, or positions corresponding thereto, which, without being bound by theory, provides excision positive and integration deficient mutations. Numbering relative to the amino acid sequence of protein of SEQ ID NO: 1, and nucleic acid sequence of SEQ ID NO: 2.
  • a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N, or positions corresponding thereto.
  • a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N and/or one or more of E284A, K286A, R287A, N310A, R333A, K334A, R336A, K349A, K350A, K368A, and K369A, or positions corresponding thereto.
  • a MLT mobile element enzyme comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, and includes one or more mutations selected from S8P and/or C13R and one of R164N, W168V, M278A, K286A, R287A, R333A, K334A, N335A, K349A, K350A, K368A, K369A, and D416N and/or one or more of E284A, K286A, R287A, N310A, R333A, K334A, R336A, K349A, K350A, K368A, and K369A and/or one R336A, or positions corresponding thereto.
  • the mobile element enzyme is or is derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, and Molossus molossus.
  • the mobile element enzyme is or is derived from any of Trichoplusia ni (SEQ ID NO: 433), Myotis myotis (SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 438, or SEQ ID NO: 439), or Pteropus vampyrus (SEQ ID NO: 434).
  • the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and/or TABLE 2B, or equivalents thereof.
  • Trichoplusia ni SEQ ID NO: 433
  • Myotis lucifugus SEQ ID NO: 437
  • Myotis myotis SEQ ID NO: 435
  • SEQ ID NO: 436 SEQ ID NO: 438
  • SEQ ID NO: 439 Pteropus vampyrus
  • Pteropus vampyrus SEQ ID NO: 434
  • Trichnoplusia ni 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61
  • the mobile element enzyme is an engineered version of a mobile element enzyme, including but not limited to monomers, dimers, tetramers, hyperactive, or Int-forms, derived from Bombyx mori, Xenopus tropicalis, or Trichoplusia ni. In embodiments, the mobile element enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, or Myotis lucifugus.
  • the mobile element enzyme is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int-), derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni or Myotis lucifugus.
  • the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and TABLE 2B, or equivalents thereof.
  • one skilled in the art can correspond such mutants to mobile element enzymes from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, Pan troglodytes, and Molossus molossus.
  • the mobile element enzyme has a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to a nucleotide sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhliim, Pan troglodytes, and Molossus molossus.
  • the mobile element enzyme has an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, and Molossus molossus. See Jebb, et al. (2020).
  • the enzyme e.g., without limitation, a mobile element enzyme
  • the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme e.g., without limitation, a mobile element enzyme
  • the enzyme is an engineered version, including but not limited to hyperactive forms, of a mobile element enzyme derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, or Homo sapiens.
  • the enzyme is either the wild type, monomer, dimer, tetramer, hyperactive, or an Int-mutant.
  • the mobile element enzymes have one or more hyperactive and/or integration deficient mutations selected from TABLE 1, TABLE 2A, and/or TABLE 2B, or equivalents thereof.
  • the mobile element enzyme has a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to a nucleotide sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, and Pan troglodytes.
  • the mobile element enzyme has an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of any of Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Molossus molossus, Pan troglodytes, and Homo sapiens.
  • the mobile element enzyme is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int-), derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pipistrellus kuhlii, Pteropus vampyrus, and Molossus molossus Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Pan troglodytes, Myotis lucifugus, and Homo sapiens.
  • a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or
  • the mobile element enzyme is either the wild type, monomer, dimer, tetramer or another multimer, hyperactive, or a an Int-mutant.
  • the mobile element enzyme is from a Tc1/mariner donor DNA system. See, e.g., Plasterk et al. Trends in Genetics.1999; 15(8):326–32.
  • the mobile element enzyme is from a Sleeping Beauty donor DNA system (see, e.g., Cell.
  • a hyperactive form of Sleeping Beauty e.g., SB100X (see Gene Therapy volume 18, pages 849–856(2011), or a piggyBac (PB) donor DNA system (see, e.g., Trends Biotechnol.2015 Sep;33(9):525-33, which is incorporated herein by reference in its entirety)
  • PB piggyBac
  • a hyperactive form of PB mobile element enzyme e.g., with seven amino acid substitutions (e.g., I30V, S103P, G165S, M282V, S509G, N570S, N538K on mPB, or functional equivalents in non-mPB, see Mol Ther Nucleic Acids.2012 Oct; 1(10): e50, which is incorporated herein by reference in its entirety); see also Yusa et al., PNAS January 25, 2011108 (4) 1531-1536; Voigt et al., PNAS January 25, 2011108 (4) 1531-1536; Voig
  • the piggyBac mobile element enzymes belong to the IS4 mobile element enzyme family. De Palmenaer et al., BMC Evolutionary Biology.2008;8:18. doi: 10.1186/1471-2148-8-18.
  • the piggyBac family includes a large diversity of donor DNAs, and any of these donor DNAs can be used in embodiments of the present disclosure. See, e.g., Bouallègue et al., Genome Biol Evol.2017;9(2):323-339.
  • the founding member of the piggyBac (super)family, insect piggyBac was originally identified in the cabbage looper moth (Trichoplusiani ni) and studied both in vivo and in vitro.
  • Insect piggyBac is known to transpose by a canonical cut-and-paste mechanism promoted by an element-encoded mobile element enzyme with a catalytic site resembling the RNase H fold shared by many recombinases.
  • the insect piggyBac donor DNA system has been shown to be highly active in a wide range of animals, including Drosophila and mice, where it has been developed as a powerful tool for gene tagging and genome engineering.
  • Other donor DNAs affiliated to the piggyBac superfamily are common in arthropods and vertebrates including Xenopus and Bombyx.
  • Mammalian piggyBac donor DNAs and mobile element enzymes including hyperactive mammalian piggyBac variants, which can be used in embodiments of the present disclosure, are described, e.g., in International Application WO2010085699, which is incorporated herein by reference in its entirety.
  • the mobile element enzyme is from a LEAP-IN 1 type or LEAP-IN donor DNA system (Biotechnol J. 2018 Oct;13(10):e1700748. doi: 10.1002/biot.201700748. Epub 2018 Jun 11).
  • the LEAPIN mobile element enzyme system includes a mobile element enzyme (e.g., without limitation, a mobile element enzyme mRNA) and a vector containing one or more genes of interest (donor DNAs), selection markers, regulatory elements, insulators, etc., flanked by the donor DNA cognate inverted terminal ends and the transposition recognition motif (TTAT).
  • a mobile element enzyme e.g., without limitation, a mobile element enzyme mRNA
  • donor DNAs genes of interest
  • selection markers e.g., selection markers, regulatory elements, insulators, etc.
  • TTAT transposition recognition motif
  • the LEAPIN mobile element enzyme generates stable transgene integrants with various advantageous characteristics, including single copy integrations at multiple genomic loci, primarily in open chromatin segments; no payload limit, so multiple independent transcriptional units may be expressed from a single construct; the integrated transgenes maintain their structural and functional integrity; and maintenance of transgene integrity ensures the desired chain ratio in every recombinant cell.
  • the mobile element enzyme is an engineered form of a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
  • PGBD1 and PGBD2 may resemble the PGBD3 donor DNA in which the mobile element enzyme ORF is flanked upstream by a 3' splice site and downstream by a polyadenylation site. See Newman et al., PLoS Genet 2008;4:e1000031. PLoS Genet 4(3): e1000031.
  • the PGBD5 inactive mobile element enzyme sequence belongs to the RNase H clan of Pfam structures, while PGBD3 has sustained only a single D to N mutation in the essential catalytic triad DDD(D) and retains the ability to bind the upstream piggyBac terminal inverted repeat. Bailey et al., DNA Repair (Amst) 2012;11:488-501.
  • the PGBD5 mobile element enzyme does not retain the catalytic DDD (D) motif found in active elements, and the mobile element enzyme is not only inactive but fails to associate with either DNA or chromatin in vivo.
  • DDD catalytic DDD
  • PGBD1 and PGBD2 are thought to be present in the common ancestor of mammals, while PGBD3 and PGBD4 are restricted to primates. See Sarkar et al., Mol Genet Genomics 2003;270:173-80.
  • the Pteropus vampyrus mobile element enzyme is closely related to PGBD4 and shares DDD catalytic domain and the C-terminal region that are involved in excision mechanisms. See Mitra et al., EMBO J 2008;27:1097-109.
  • a mammalian mobile element enzyme which has gene cleavage and/or gene integration activity, can be constructed based on alignment of the amino acid sequence of Pteropus vampyrus mobile element enzyme to PGBD1, PGBD2, PGBD3, PGBD4, and PGBD5 sequences. Also, in embodiments. the mammalian mobile element enzyme has mutations that confers hyperactivity to a recombinant mammalian mobile element enzyme.
  • the mobile element enzyme has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
  • the mobile element enzyme has gene cleavage activity (Exc+) and/or lacks gene integration activity (Int-).
  • an enzyme capable of targeted genomic integration is a recombinant mammalian mobile element enzyme that was derived by, in part, aligning several inactive mobile element enzyme sequences from a human genome to Pteropus vampyrus mobile element enzyme sequence.
  • the Pteropus vampyrus mobile element enzyme has an amino acid sequence having at least 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to SEQ ID NO: 430 (or a functional equivalent thereof.
  • the Pteropus vampyrus mobile element enzyme has an amino acid sequence of SEQ ID NO: 430, or a functional equivalent thereof.
  • the Pteropus vampyrus mobile element enzyme has a nucleotide sequence having at least 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to SEQ ID NO: 429 or a codon-optimized variant thereof.
  • the mobile element enzyme is a mammalian mobile element enzyme, such as a mobile element enzyme from a bat, e.g., without limitation, Pteropus vampyrus.
  • the mobile element enzyme is an engineered form that is based on a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
  • the mobile element enzyme includes but is not limited to an engineered version that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int-), of an engineered version of a mobile element enzyme reconstructed from Homo sapiens or a predecessor thereof.
  • the mobile element enzyme is an engineered form that is based on a mobile element enzyme reconstructed from mammalian species.
  • the mobile element enzyme includes but is not limited to an engineered that is a monomer, dimer, tetramer (or another multimer), hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int-), of a mobile element enzyme reconstructed from mammalian species.
  • the donor DNA is included in a vector comprising left and right end sequences recognized by the mobile element enzyme.
  • the end sequences are selected from MER, MER75A, MER75B, and MER85.
  • the end sequences are selected from nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 441, and SEQ ID NO: 22, or a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) thereto.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity
  • one or more of the end sequences are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) identity to the nucleotide sequence of SEQ ID NO: 12, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 12 is positioned at the 5’ end of the donor DNA.
  • the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 17, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 17 is positioned at the 3’ end of the donor DNA.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least
  • the end sequences which can be from, e.g., Pteropus vampyrus, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 13, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 13 is positioned at the 5’ end of the donor DNA.
  • the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18 is positioned at the 3’ end of the donor DNA.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at
  • the end sequences which can be, e.g., PGBD4, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 14, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 14 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 18, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 19 is positioned at the 3’ end of the donor DNA.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about
  • the end sequences which can be, e.g., MER75, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 15, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 15 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 20, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity(e.g. a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 20 is positioned at the 3’ end of the donor DNA.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99%
  • the end sequences which can be, e.g., MER75B, are optionally flanked by a TTAA (SEQ ID NO: 440) sequence.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) identity to the nucleotide sequence of SEQ ID NO: 16, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 16 is positioned at the 5’ end of the donor DNA.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity (e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) to the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 441 is positioned at the 3’ end of the donor DNA.
  • a nucleotide sequence having at least about 90% identity e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least
  • a donor DNA is or comprises a vector comprising a donor DNA comprising one or more end sequences recognized by an enzyme such as, for example a mobile element enzyme.
  • the end sequences are selected from Pteropus vampyrus, MER75, MER75A, and MER75B. MERs contain end sequences with similarity to piggyBac-like mobile elements and exhibit duplications of their presumed TTAA (SEQ ID NO: 440) target sites.
  • the end sequences are selected from nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 441, and SEQ ID NO: 22, or a nucleotide sequence having at least about 90% identity (e.g. a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity) thereto.
  • a nucleotide sequence having at least about 90% identity e.g. a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity
  • the mobile element enzyme has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the mobile element enzyme has an amino acid sequence having S8P, G17R, and/or K134K mutation relative to the amino acid sequence of SEQ ID NO: 4 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having S8P, G17R, and/or K134K mutation relative to the amino acid sequence of SEQ ID NO: 5 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having I83P and/or V118R mutation relative to the amino acid sequence of SEQ ID NO: 6 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having S20P and/or A29R mutation relative to the amino acid sequence of SEQ ID NO: 7 or a functional equivalent thereof.
  • the mobile element enzyme has an amino acid sequence having T4P and/or L13R mutation relative to the amino acid sequence of SEQ ID NO: 8 or a functional equivalent thereof. In embodiments, the mobile element enzyme has an amino acid sequence having A12P and/or I28R mutation and/or R152K mutation relative to the amino acid sequence of SEQ ID NO: 9 or a functional equivalent thereof.
  • the enzyme capable of targeted genomic integration e.g., without limitations, a mobile element enzyme
  • the enzyme capable of targeted genomic integration e.g., without limitations, a mobile element enzyme
  • the enzyme e.g., without limitation, a mobile element enzyme
  • the enzyme is an engineered version, including but not limited to a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or has a reduced interaction with non-TTAA (SEQ ID NO: 440) recognitions sites (Int-), and is derived from any of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens.
  • a mobile element enzyme that is a monomer, dimer, tetramer, hyperactive, or has a reduced interaction with non-TTAA (SEQ ID
  • the mobile element enzyme is either the wild type, monomer, dimer, tetramer or another multimer, hyperactive, or an Int-mutant.
  • Targeting Chimeric Constructs e.g., in embodiments, the enzyme, without limitation, a mobile element enzyme, comprises a targeting element.
  • the targeting element is or comprises a gene-editing system, e.g., that is catalytically inactive (or “dead”).
  • the enzyme without limitation, a mobile element enzyme, associated with the targeting element, is capable of inserting the donor DNA (e.g., mobile element) comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a GSHS.
  • the enzyme without limitation, a mobile element enzyme, associated with the targeting element has one or more mutations which confer hyperactivity.
  • the enzyme, without limitation, a mobile element enzyme, associated with the targeting element has gene cleavage activity (Exc+) and/or gene integration activity (Int+).
  • the enzyme without limitation, a mobile element enzyme, associated with the targeting element has gene cleavage activity (Exc+) and/or a lack of gene integration activity (Int-).
  • the targeting element comprises one or more proteins or nucleic acids that are capable of binding to a nucleic acid.
  • the targeting element comprises one or more of a of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, paternally expressed gene 10 (PEG10), and TnsD.
  • the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD).
  • the TALE DBD comprises one or more repeat sequences.
  • the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids.
  • the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.
  • RVD recognizes one base pair in the nucleic acid molecule.
  • the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI.
  • the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.
  • AAVS1 adeno-associated virus site 1
  • C-C motif chemokine receptor 5
  • the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the targeting element comprises a Cas9 enzyme guide RNA complex.
  • the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.
  • the targeting element comprises a Cas12 enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex.
  • the targeting element comprises a Cas12k enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex.
  • a targeting chimeric system or construct having a DBD fused to a mobile element enzyme, directs binding of an enzyme capable of targeted genomic integration (e.g., without limitation, a mobile element enzyme) to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di-residues (RVD) or gRNA) near an enzyme recognition site.
  • TALE transcription activator-like effector proteins
  • RVD repeat variable di-residues
  • gRNA binds to human GSHS.
  • dCas9 i.e., deficient for nuclease activity
  • gRNAs directed to bind at a desired sequence of DNA in GSHS.
  • TALEs described herein can physically sequester the enzyme such as, e.g., a mobile element enzyme, to GSHS and promote transposition to nearby TTAA (SEQ ID NO: 440) sequences in close proximity to the RVD TALE nucleotide sequences.
  • GSHS in open chromatin sites are specifically targeted based on the predilection for mobile element enzymes to insert into open chromatin.
  • an enzyme capable of targeted genomic integration e.g., without limitation, a recombinase, integrase, or a mobile element enzyme such as, without limitation, a mammalian mobile element enzyme
  • a TALE DNA binding domain DBD
  • a Cas-based gene-editing system such as, e.g., Cas9 or a variant thereof.
  • the targeting element targets the enzyme to a locus of interest.
  • the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof.
  • a CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity.
  • sgRNA single-guide RNA
  • the inactivated form of Cas9 which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity.
  • dCas9 has no substantial nuclease activity.
  • CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences.
  • gRNA guide RNA
  • dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome.
  • gRNA guide RNA
  • dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome.
  • the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9” ) guide RNA complex.
  • the dCas9/gRNA complex comprises a guide RNA selected from: GTTTAGCTCACCCGTGAGCC (SEQ ID NO: 91), CCCAATATTATTGTTCTCTG (SEQ ID NO: 92), GGGGTGGGATAGGGGATACG (SEQ ID NO: 93), GGATCCCCCTCTACATTTAA (SEQ ID NO: 94), GTGATCTTGTACAAATCATT (SEQ ID NO: 95), CTACACAGAATCTGTTAGAA (SEQ ID NO: 96), TAAGCTAGAGAATAGATCTC (SEQ ID NO: 97), and TCAATACACTTAATGATTTA (SEQ ID NO: 98), wherein the guide RNA directs the enzyme to a chemokine (C-C motif) receptor 5 (CCR5) gene.
  • C-C motif chemokine receptor 5
  • the dCas9/gRNA complex comprises a guide RNA selected from: CACCGGGAGCCACGAAAACAGATCC (SEQ ID NO: 99);CACCGCGAAAACAGATCCAGGGACA (SEQ ID NO: 100); CACCGAGATCCAGGGACACGGTGCT (SEQ ID NO: 101); CACCGGACACGGTGCTAGGACAGTG (SEQ ID NO: 102); CACCGGAAAATGACCCAACAGCCTC (SEQ ID NO: 103); CACCGGCCTGGCCGGCCTGACCACT (SEQ ID NO: 104); CACCGCTGAGCACTGAAGGCCTGGC (SEQ ID NO: 105); CACCGTGGTTTCCACTGAGCACTGA (SEQ ID NO: 106); CACCGGATAGCCAGGAGTCCTTTCG (SEQ ID NO: 107); CACCGGCGCTTCCAGTGCTCAGACT (SEQ ID NO: 108); CACCGCAGTGCTCAGACTAGGGAAG (SEQ ID NO: 109
  • the guide RNAs are: AATCGAGAAGCGACTCGACA (SEQ ID NO: 425), and tgccctgcaggggagtgagc (SEQ ID NO: 426).
  • the guide RNAs are gaagcgactcgacatggagg (SEQ ID NO: 427) and cctgcaggggagtgagcagc (SEQ ID NO: 428).
  • guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 3A-3F.
  • guide RNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 3A: In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to the TTAA site in hROSA26 (e.g., hg38 chr3:9,396,133-9,396,305) are shown in TABLE 3B: In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, to the AAVS1 (e.g., hg38 chr19:55,112,851-55,113,324) are shown in TABLE 3C: In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-
  • a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k).
  • the targeting element comprises a Cas12 enzyme guide RNA complex.
  • the targeting element is selected from a zinc finger (ZF), catalytically inactive Zinc finger, transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)- associated protein, any of which are, in embodiments, catalytically inactive.
  • ZF zinc finger
  • TALE transcription activator-like effector
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof.
  • the CRISPR-associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof.
  • the mobile element enzyme is capable of inserting a donor DNA at a TA dinucleotide site or a TTAA tetranucleotide site in a GSHS of a nucleic acid molecule.
  • the mobile element enzyme is suitable for causing insertion of the donor DNA in a GSHS when contacted with a biological cell.
  • the targeting element is suitable for directing the mobile element enzyme to the GSHS sequence.
  • the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD).
  • TALE DBD comprises one or more repeat sequences.
  • the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids.
  • the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.
  • the targeting element e.g., TALE or Cas (e.g., Cas9 or Cas12, or variants thereof) DBDs cause the mammalian mobile element enzyme to bind specifically to human GSHS.
  • the TALEs or Cas DBDs sequester the mobile element enzyme to GSHS and promote transposition to nearby TA dinucleotide or a TTAA tetranucleotide sites which can be located in proximity to the repeat variable di-residues (RVD) TALE or gRNA nucleotide sequences.
  • the GSHS regions are located in open chromatin sites that are susceptible to mobile element enzyme activity. Accordingly, the mammalian mobile element enzyme does not only operate based on its ability to recognize TA or TTAA sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Cas DBD.
  • the chimeric mobile element enzyme in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies.
  • a chimeric mobile element enzyme is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Cas DBD, for transposition.
  • a DBD fused thereto such as a TALE or Cas DBD
  • the described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk.
  • the described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies.
  • TALE or Cas DBDs are customizable, such as a TALE or Cas DBDs is selected for targeting a specific genomic location.
  • the genomic location is in proximity to a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site.
  • TTAA SEQ ID NO: 440
  • TALE repeat sequences e.g., modular arrays
  • gRNA e.g., gRNA which are linked together to recognize flanking DNA sequences.
  • TALE or gRNA can recognize certain base pair(s) or residue(s).
  • TALE nucleases TALENs
  • TALENs are a known tool for genome editing and introducing targeted double-stranded breaks. TALENs comprise endonucleases, such as FokI nuclease domain, fused to a customizable DBD. This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells.
  • the DBD includes a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DBDs by selecting a combination of repeat segments containing the appropriate RVDs. Boch et al. Nature Biotechnology.2011; 29 (2): 135–6. Accordingly, TALENs can be readily designed using a “protein-DNA code” that relates modular DNA-binding TALE repeat domains to individual bases in a target-binding site. See Joung et al. Nat Rev Mol Cell Biol.2013;14(1):49-55.
  • the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids.
  • the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids.
  • the RVD can recognize certain base pair(s) or residue(s).
  • the RVD recognizes one base pair in the nucleic acid molecule.
  • the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI.
  • the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA.
  • the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.
  • the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor; and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the GSHS comprises one or more of TGGCCGGCCTGACCACTGG (SEQ ID NO: 23), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25), TCCACTGAGCACTGAAGGC (SEQ ID NO: 26), TGGTTTCCACTGAGCACTG (SEQ ID NO: 27), TGGGGAAAATGACCCAACA (SEQ ID NO: 28), TAGGACAGTGGGGAAAATG (SEQ ID NO: 29), TCCAGGGACACGGTGCTAG (SEQ ID NO: 30), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 31), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 32), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 33), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 34), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 35), TGGTTTCCGAGCTTGACCC (SEQ ID
  • the TALE DBD binds to one of TGGCCGGCCTGACCACTGG (SEQ ID NO: 23), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25), TCCACTGAGCACTGAAGGC (SEQ ID NO: 26), TGGTTTCCACTGAGCACTG (SEQ ID NO: 27), TGGGGAAAATGACCCAACA (SEQ ID NO: 28), TAGGACAGTGGGGAAAATG (SEQ ID NO: 29), TCCAGGGACACGGTGCTAG (SEQ ID NO: 30), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 31), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 32), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 33), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 34), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 35), TGGTTTCCGAGCTTGACC
  • the TALE DBD comprises one or more of: NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG NH NH, NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD NG NH, NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD, NH NI HD NG NH NI NH HD NI HD NG NH NI NI NH NH HD, NH NH NG NG NG HD HD NI HD NG NH NI NI HD NG NH, NH NH NH NH NH NI NI NI NI HD NG NH, NH NH NH NH NI NI NI NI NG NH NI HD NI HD NG NH, NH NH NH NH NI NI NI NI NG NH NI HD NI HD NG NH, NH NH
  • the TALE DBD comprises one or more of the sequences outlined herein or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the GSHS and the TALE DBD sequences are selected from: TGGCCGGCCTGACCACTGG (SEQ ID NO: 23) and NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG NH NH; TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24) and NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH; TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25) and NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD; TCCACTGAGCACTGAAGGC (SEQ ID NO: 26) and HD HD NI HD NG NH NI NH HD NI HD NG NH NI NI NH NH HD; TGGTTTCCACTGAGCACTG (SEQ ID NO: 27) and NH NH NG NG NG HD HD NI HD NG NH NH
  • the GSHS is within about 25, or about 50, or about 100, or about 150, or about 200, or about 300, or about 500 nucleotides of the TA dinucleotide site or TTAA (SEQ ID NO: 440) tetranucleotide site.
  • Illustrative DNA binding codes for human genomic safe harbor in areas of open chromatin via TALEs, encompassed by various embodiments are provided in TABLE 4A-4F.
  • TALEs encompassed by various embodiments are provided in TABLE 4A-4F, e.g., having a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to any of the sequences in TABLE 4A-4F.
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to the TTAA site in hROSA26 e.g., hg38 chr3:9,396,133-9,396,305
  • TABLE 4B TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to the AAVS1 (e.g., hg38 chr19:55,112,851-55,113,324) are shown in TABLE 4C:
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 4 are shown in TABLE 4D:
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 22 are shown in TABLE 4E:
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome X are shown in TABLE 4D:
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 22 are shown in embodiments.
  • TALEs for targeting human genomic safe harbor sites using any of the TALE-based targeting elements to Chromosome 22 are shown in TABLE 4E:
  • the mobile element enzyme is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site.
  • TTAA SEQ ID NO: 440
  • Illustrative DNA binding codes for human genomic safe harbor in areas of open chromatin via ZNFs encompassed by various embodiments are provided in TABLE 5A-5E.
  • there is provided a variant of the ZNFs, encompassed by various embodiments are provided in TABLE 5A-5E, e.g., having a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to any of the sequences in TABLE 5A-5E.
  • ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to the TTAA site in hROSA26 are shown in TABLE 5A:
  • ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to the AAVS1 are shown in TABLE 5B:
  • ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome 4 are shown in TABLE 5C:
  • ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome 22 are shown in TABLE 5D:
  • ZNFs for targeting human genomic safe harbor sites using any of the ZNF-based targeting elements to Chromosome X are shown in TABLE 5E:
  • the mobile element enzyme is capable of inserting a donor DNA at a TA dinucleotide site.
  • the mobile element enzyme is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site.
  • the present disclosure relates to a system having nucleic acids encoding the enzyme and the donor DNA, respectively.
  • FIGs.1A-1D show examples of a system in accordance with embodiments of the present disclosure.
  • Linkers In embodiments, the targeting element comprises a nucleic acid binding component of the gene-editing system (targeting element).
  • the enzyme capable of targeted genomic integration e.g., without limitation, a chimeric mobile element enzyme
  • the targeting element e.g., nucleic acid binding component of the gene-editing system are fused or linked to one another.
  • the mobile element enzyme and the targeting element are fused or linked to one another.
  • the mobile element enzyme and the targeting element e.g., nucleic acid binding component of the gene-editing system are connected via a linker.
  • the linker is a flexible linker.
  • the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is from about 1 to about 12.
  • the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.
  • the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt.
  • the mobile element enzyme and the targeting element are encoded on a single polypeptide.
  • the donor DNA comprises a gene encoding a complete polypeptide.
  • the donor DNA comprises a gene which is defective or substantially absent in a disease state.
  • Inteins Inteins are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Current Protein & Peptide Science, 20(5), 408–424, which is incorporated by reference herein in its entirety.
  • Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein.
  • Inteins apply standard enzymatic strategies to excise themselves post-translationally from a precursor protein via protein splicing. Nanda et al., Microorganisms vol. 8,12 2004. 16 Dec. 2020, doi:10.3390/microorganisms8122004.
  • An intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence. For example, split inteins have been used to control the delivery of heterologous genes into transgenic organisms.
  • intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, or TALEs, allows creation of a split-enzyme system such as, without limitation, split-MLT mobile element enzyme system, that permits reconstitution of the full-length enzyme, e.g., MLT mobile element enzyme, from two smaller fragments.
  • a nucleic acid encoding the enzyme capable of targeted genomic integration comprises an intein.
  • the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme.
  • an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Patent No.9,200,045; Mootz et al., J. Am. Chem. Soc.2002; 124, 9044-9045; Mootz et al., J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., Proc. Natl.
  • the intein is NpuN (Intein-N) (SEQ ID NO: 423) and/or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • SEQ ID NO: 423 nucleotide sequence of NpuN (Intein-N) GGCGGATCTGGCGGTAGTGCTGAGTATTGTCTGAGTTACGAAACGGAAATACTCACGGTTGAGTATGGGCTTCTTCC AATTGGCAAAATCGTTGAAAAGCGCATAGAGTGTACGGTGTATTCCGTCGATAACAACGGTAATATCTACACCCAGC CGGTAGCTCAGTGGCACGACCGAGGCGAACAGGAAGTGTTCGAGTATTGCTTGGAAGATGGCTCCCTTATCCGCGCC ACTAAAGACCATAAGTTTATGACGGTTGACGGGCAGATGCTGCCTATAGACGAAATATTTGAGAGAGCTGGACTT GATGAGAGTCGATAATCTGCCAAAT SEQ ID NO: 424: nucleotide sequence of NpuC (Intein-C) GGCGGATCTGGCGGTAGTGGGGGTTCCGGATCCATAAAGATAGCTACTAGGAAATATCTTGGCAAACAAAACGTCTA TGACAAA
  • a nucleic acid encoding the transgene is DNA.
  • the enzyme e.g., without limitation, the mobile element enzyme
  • the nucleic acid is RNA, optionally a helper RNA.
  • the nucleic acid is RNA that has a 5’-m7G cap (cap0, or cap1, or cap2), optionally with pseudouridine substitution (e.g., without limitation n-methyl-pseudouridine), and optionally a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.
  • the poly-A tail is of about 30 nucleotides in length, optionally 34 nucleotides in length.
  • a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus.
  • the nucleic acid that is RNA has a 5’-m7G cap (cap 0, or cap 1, or cap 2).
  • the nucleic acid comprises a 5' cap structure, a 5'-UTR comprising a Kozak consensus sequence, a 5'-UTR comprising a sequence that increases RNA stability in vivo, a 3'-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3' poly(A) tail.
  • the enzyme e.g., without limitation, a mobile element enzyme
  • the vector is a non-viral vector.
  • a nucleic acid encoding the enzyme in accordance with embodiments of the present disclosure is DNA.
  • a construct comprising a donor DNA is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector.
  • the construct is DNA, which is referred to herein as a donor DNA.
  • sequences of a nucleic acid encoding the donor DNA is codon optimized to provide improved mRNA stability and protein expression in mammalian systems.
  • the enzyme and the donor DNA are included in different vectors. In embodiments, the enzyme and the donor DNA are included in the same vector.
  • a nucleic acid encoding the enzyme capable of targeted genomic integration e.g., without limitation, a mobile element enzyme which is a chimeric mobile element enzyme
  • RNA e.g., helper RNA
  • a nucleic acid encoding a donor DNA is DNA.
  • a donor DNA often includes an open reading frame that encodes a transgene at the middle of donor DNA and terminal repeat sequences at the 5’ and 3’ end of the donor DNA. The translated mobile element enzyme binds to the 5’ and 3’ sequence of the donor DNA and carries out the transposition function.
  • donor DNA or transgene are used interchangeably with mobile elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides.
  • the term donor DNA is well known to those skilled in the art and includes classes of donor DNAs that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and/or directly repeated long terminal repeats (LTRs) at the ends.
  • LTRs long terminal repeats
  • the donor DNA as described herein may be described as a piggyBac like element, e.g., a donor DNA element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor DNA.
  • the donor DNA is flanked by one or more end sequences or terminal ends.
  • the donor DNA is or comprises a gene encoding a complete polypeptide.
  • the donor DNA is or comprises a gene which is defective or substantially absent in a disease state.
  • the donor DNA includes a MLT mobile element enzyme (e.g., without limitation, a MLT mobile element enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11).
  • the mobile element enzyme can act on a left terminal end having a nucleotide sequence of SEQ ID NO: 431 or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the donor DNA can act on a right terminal end having a nucleotide sequence of SEQ ID NO: 432 or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the donor DNA acts on both MLT left donor DNA end and MLT right donor DNA end, having nucleotide sequences of SEQ ID NO: 431 and of SEQ ID NO: 432 respectively, or a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • a MLT left donor DNA end is as follows TTAACACTTGGATTGCGGGAAACGAGTTAAGTCGGCTCGCGTGAATTGCGCGTACTCCGCGGGAGCCGTC TTAACTCGGTTCATATAGATTTGCGGTGGAGTGCGGGAAACGTGTAAACTCGGGCCGATTGTAACTGCGT ATTACCAAATATTTGTT (SEQ ID NO: 431)
  • a MLT right donor DNA end is as follows AATTATTTATGTACTGAATAGATAAAAAAATGTCTGTGATTGAATAAATTTTCATTTTACACAAGAAA CCGAAAATTTCATTTCAATCGAACCCATACTTCAAAAGATATAGGCATTTTAAACTAACTCTGATTTTGC GCGGGAAACCTAAATAATTGCCCGCGCCATCTTATATTTTGGCGGGAAATTCACCCGACACCGTGGTGTT AA (SEQ ID NO: 432).
  • a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene.
  • a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes.
  • the insulators flank the donor DNA (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences.
  • the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5’ -HS4 chicken ⁇ -globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo-Humeral Dystrophy (FSHD).
  • the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety.
  • the transgene is inserted into a GSHS location in a host genome.
  • GSHSs is defined as loci well-suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto-oncogene activation, tumor suppressor inactivation, or insertional mutagenesis.
  • GSHSs can defined by the following criteria: (1) distance of at least 50 kb from the 5’ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat Biotechnol 2011;29:73-8; Bejerano et al. Science 2004;304:1321-5.
  • CCR5 chemokine C-C motif receptor 5
  • a homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans.
  • Disrupted CCR5 expression naturally occurring in about 1% of the Caucasian population, does not appear to result in any reduction in immunity.
  • a clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases.
  • the donor DNA is under control of a tissue-specific promoter.
  • the tissue-specific promoter is, e.g., without limitation, a liver-specific promoter.
  • the liver-specific promoter is an LP1 promoter that, in embodiments, is a human LP1 promoter.
  • the LP1 promoter is described, e.g., in Nathwani et al. Blood vol. 2006;107(7):2653-61, and it is constructed, without limitation, as described in Nathawani et al.
  • the present nucleic acids include polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs or derivatives thereof.
  • transcriptionally- activated polynucleotides such as methylated or capped polynucleotides are provided.
  • the present compositions are mRNA or DNA.
  • the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide.
  • the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences.
  • Such vectors may include, among others, chromosomal and episomal vectors, e.g., vectors bacterial plasmids, from donor DNAs, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors from combinations thereof.
  • the present constructs may contain control regions that regulate as well as engender expression.
  • the construct comprising the enzyme and/or transgene is codon optimized.
  • Transgene codon optimization is used to optimize therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in a host organism or cell. Codon optimization methods are known in the art and described in, for example, WO 2007/142954, which is incorporated by reference herein in its entirety. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases.
  • the construct comprising the enzyme and/or transgene includes several other regulatory elements that are selected to ensure stable expression of the construct.
  • the non-viral vector is a DNA plasmid that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes.
  • the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5'-HS4 chicken ⁇ - globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo- Humeral Dystrophy (FSHD).
  • the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al.
  • the gene of the construct comprising the enzyme and/or transgene is capable of transposition in the presence of a mobile element enzyme.
  • the non-viral vector in accordance with embodiments of the present disclosure comprises a nucleic acid construct encoding a mobile element enzyme.
  • the mobile element enzyme is an RNA mobile element enzyme plasmid.
  • the non-viral vector further comprises a nucleic acid construct encoding a DNA mobile element enzyme plasmid.
  • the mobile element enzyme is an in vitro-transcribed mRNA mobile element enzyme.
  • the mobile element enzyme is capable of excising and/or transposing the gene from the construct comprising the enzyme and/or transgene to site- or locus-specific genomic regions.
  • the enzyme and the donor DNA are included in the same vector.
  • the enzyme is disposed on the same (cis) or different vector (trans) than a donor DNA with a transgene. Accordingly, in embodiments, the enzyme and the donor DNA encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the enzyme and the donor DNA encompassing a transgene are in trans configuration such that they are included in different vectors.
  • the vector is any non-viral vector in accordance with the present disclosure.
  • a nucleic acid encoding the enzyme capable of targeted genomic integration in accordance with embodiments of the present disclosure is provided.
  • the nucleic acid is or comprises DNA or RNA.
  • the nucleic acid encoding the enzyme is DNA.
  • the nucleic acid encoding the enzyme capable of targeted genomic integration e.g., a chimeric mobile element enzyme
  • RNA such as, e.g., helper RNA.
  • the chimeric mobile element enzyme is incorporated into a vector.
  • the vector is a non-viral vector.
  • a nucleic acid encoding the transgene in accordance with embodiments of the present disclosure is provided.
  • the nucleic acid is or comprises DNA or RNA.
  • the nucleic acid encoding the transgene is DNA.
  • the nucleic acid encoding the e transgene is RNA such as, e.g., helper RNA.
  • the transgene is incorporated into a vector.
  • the vector is a non-viral vector.
  • the present enzyme can be in the form or an RNA or DNA and have one or two N-terminus nuclear localization signal (NLS) to shuttle the protein more efficiently into the nucleus.
  • NLS nuclear localization signal
  • the present enzyme further comprises one, two, three, four, five, or more NLSs.
  • NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 348).
  • the NLS comprises the consensus sequence (K/R)(K/R)X 10-12 (K/R) 3/5 (SEQ ID NO: 349), where (K/R) 3/5 represents at least three of the five amino acids is either lysine or arginine.
  • the NLS comprises the c-myc NLS.
  • the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 350).
  • the NLS is the nucleoplasmin NLS.
  • the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 351).
  • the NLS comprises the SV40 Large T-antigen NLS.
  • the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 352).
  • the NLS comprises three SV40 Large T-antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 353).
  • the NLS may comprise mutations/variations in the above sequences such that they contain 1 or more substitutions, additions or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).
  • a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided.
  • Lipids In embodiments, at least one of the first nucleic acid and the second nucleic acid is in the form of a lipid nanoparticle (LNP). In embodiments, a composition comprising the first and second nucleic acids is in the form of an LNP.
  • a nucleic acid encoding the enzyme and a nucleic acid encoding the transgene are contained within the same lipid nanoparticle (LNP).
  • the nucleic acid encoding the enzyme and the nucleic acid encoding the donor DNA are a mixture incorporated into or associated with the same LNP.
  • the nucleic acid encoding the enzyme and the nucleic acid encoding the donor DNA are in the form of a co-formulation incorporated into or associated with the same LNP.
  • the LNP is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol – 2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol- 3phosphocholine (DSPC) and/or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).
  • DOTAP 1,2-di
  • an LNP is as described, e.g., in Patel et al., J Control Release 2019; 303:91-100.
  • the LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc).
  • a nanoparticle is a particle having a diameter of less than about 1000 nm.
  • nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm.
  • a cell in accordance with the present disclosure is prepared via an in vivo genetic modification method.
  • a genetic modification in accordance with the present disclosure is performed via an ex vivo method.
  • a cell in accordance with the present disclosure is prepared by contacting the cell with an enzyme capable of targeted genomic integration (e.g., without limitation, a mammalian mobile element enzyme) in vivo.
  • the cell is contacted with the enzyme ex vivo.
  • the present method provides reduced insertional mutagenesis or oncogenesis as compared to a method with a non-chimeric mobile element enzyme.
  • the transgene of interest in accordance with embodiments of the present disclosure can encode various genes.
  • the enzyme (e.g., without limitations, a mobile element enzyme), and the donor DNA are included in the same pharmaceutical composition. In embodiments, the enzyme (e.g., without limitations, a mobile element enzyme) and the donor DNA are included in different pharmaceutical compositions. In embodiments, the enzyme and the donor DNA are co-transfected. In embodiments, the enzyme and the donor DNA are transfected separately. In embodiments, a method of producing a molecule with the transfected cell is provided, wherein the transfected cell is generated using a method of making a viral packaging or producer cell line in accordance with embodiments of the present disclosure.
  • the molecule is optionally a protein. In embodiments, the protein is an antibody.
  • the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with REP expression or substantially enhanced REP expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression and REP expression or substantially enhanced REP expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with REP expression or substantially enhanced VP1 expression. In embodiments, the method of making a viral packaging or producer cell line of the present disclosure provides a stable cell with substantially reduced or ablated CAP expression.
  • the viral packaging or producer cell line of the present disclosure is suitable for providing substantially reduced empty or cargo-free capsid.
  • a transfected cell for gene therapy is provided, wherein the transfected cell is generated using a method of making a viral packaging or producer cell line in accordance with embodiments of the present disclosure.
  • a method of delivering a cell therapy is provided, comprising administering to a patient in need thereof the transfected cell generated using a method of making a viral packaging or producer cell line in accordance with embodiments of the present disclosure.
  • a method of treating a disease or condition using a cell therapy comprising administering to a patient in need thereof the transfected cell generated using a method of making a viral packaging or producer cell line in accordance with embodiments of the present disclosure.
  • a method of treating a disease or condition using an antibody therapy comprising administering to a patient in need thereof the transfected cell generated using a method of making a viral packaging or producer cell line in accordance with embodiments of the present disclosure.
  • the disease or condition is or comprises cancer.
  • the cancer is or comprises an adrenal cancer, a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, a cancer of the esophagus, a gastric cancer, a head/neck cancer, a hepatobiliary cancer, a kidney cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a pelvis cancer, a pleura cancer, a prostate cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a uterine cancer, a lymphoma, a melanoma, a multiple myeloma, or a leukemia.
  • an adrenal cancer a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer
  • the cancer is selected from one or more of the basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular
  • the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
  • the disease or condition is or comprises an infectious disease.
  • the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof.
  • the infectious disease is or comprises a disease comprising a viral infection, a parasitic infection, or a bacterial infection.
  • the viral infection is caused by a virus of family Flaviviridae, a virus of family Picornaviridae, a virus of family Orthomyxoviridae, a virus of family Coronaviridae, a virus of family Retroviridae, a virus of family Paramyxoviridae, a virus of family Bunyaviridae, or a virus of family Reoviridae.
  • the virus of family Coronaviridae comprises a betacoronavirus or an alphacoronavirus, optionally wherein the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43, or the alphacoronavirus is selected from a HCoV-NL63 and HCoV-229E.
  • the infectious disease comprises a coronavirus infection 2019 (COVID-19).
  • the method requires a single administration.
  • the method requires a plurality of administrations.
  • the present disclosure provides an ex vivo gene therapy approach.
  • the method that is used to treat an inherited or acquired disease in a patient in need thereof comprises (a) contacting a cell obtained from a patient (autologous) or another individual (allogeneic) with a transfected cell in accordance with embodiments of the present disclosure; and (b) administering the cell to a patient in need thereof.
  • One of the advantages of ex vivo gene therapy is the ability to “sample” the transduced cells before patient administration. This facilitates efficacy and allows performing safety checks before introducing the cell(s) to the patient. For example, the transduction efficiency and/or the clonality of integration can be assessed before infusion of the product.
  • a composition comprising transfected cells in accordance with the present disclosure comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile, and the fluid should be easy to draw up by a syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer- sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid.
  • PCPP-SA poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid]
  • FAD-SA fatty acid dimer- sebacic acid
  • polyglycolic acid collagen
  • polyorthoesters polyethyleneglycol-coated liposomes
  • polylactic acid polylactic acid
  • the stable integration comprises an introduction of a polynucleotide into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome.
  • a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure.
  • the organism may be a mammal or an insect.
  • the organism When the organism is a mammal, the organism may include, but is not limited to, a mouse, a rat, a monkey, a dog, a rabbit, and the like. When the organism is an insect, the organism may include, but is not limited to, a fruit fly, a mosquito, a bollworm, and the like.
  • the cells produced in accordance with embodiments of the present disclosure, and/or components for generating cells is included in a container, kit, pack, or dispenser together with instructions for administration. Also provided herein are kits comprising: one or more genetic constructs encoding the present enzyme and donor DNA and instructions and/or reagents for the use of the same.
  • kits comprising: i) a transfected cell in accordance with embodiments of the present disclosure, ii) instructions for the use of the transfected cell.
  • a kit is provided for creating an AAV of packaging cell line or an AAV library of packaging cell lines, and instructions for creating a library. A subsequent user-defined transfection of the viral genome can be performed to generate a producer cell line.
  • the kit includes instructions for transfection of an AAV packaging cell line with a desired transgene.
  • kits includes an AAV library of packaging cell lines and instructions for creating a library
  • a kit is provided that comprises an enzyme (e.g., without limitation, a recombinant mammalian mobile element enzyme) or a nucleic acid in accordance with embodiments of the present disclosure, and instructions for introducing DNA and/or RNA into a cell using the enzyme.
  • an enzyme e.g., without limitation, a recombinant mammalian mobile element enzyme
  • nucleic acid in accordance with embodiments of the present disclosure
  • instructions for introducing DNA and/or RNA into a cell using the enzyme e.g., without limitation, a recombinant mammalian mobile element enzyme
  • an “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
  • the term “in vivo” refers to an event that takes place in a subject’s body.
  • the term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject’s body.
  • the cell, tissue and/or organ may be returned to the subject’s body in a method of treatment or surgery.
  • the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions.
  • the variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
  • Carrier or “vehicle” as used herein refer to carrier materials suitable for drug administration.
  • Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid, or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients.
  • pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose.
  • the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • an effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to about 50% of the population) and the ED 50 (the dose therapeutically effective in about 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 /ED 50 .
  • compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.
  • the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • SEQ ID NO: 1 MLT mobile element enzyme protein (amino acid sequence of a variant of the hyperactive mobile element enzyme with S at position 8 and C at position 13 (572 amino acids) MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEWSHV DNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDITPQEMKK FLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKVRPVLDYFVP KFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGEGKRLLETIQTVV SPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFIRKNDILL
  • FIG.2E illustrates a non-limiting example of a system comprising a nucleic acid (e.g., helper RNA) encoding an enzyme capable of performing targeted genomic integration and a nucleic acid encoding a mobile element enzyme (donor DNA).
  • a nucleic acid e.g., helper RNA
  • the helper RNA is translated into a bioengineered enzyme (e.g., integrase, recombinase, or mobile element enzyme) that recognizes specific ends and seamlessly inserts the donor DNA into the human genome in a site-specific manner without a footprint.
  • enzymes e.g., chimeric mobile element enzymes
  • FIGs.2A-D depict representations of chimeric mobile element enzyme designed using human GSHS TALE or Cas9/gRNA DBD.
  • FIG.2A. TALEs includes nuclear localization signals (NLS) and an activation domain (AD) to function as transcriptional activators.
  • a central tandem repeat domain confers specific DNA-binding and host specificity.
  • Translocation signal (TD) and four cryptic repeats required for initiation of DNA binding and for the recognition of 5’ -T 0 are located at the N-terminus (checkered rectangles).
  • Each 34 amino acid (aa) long repeat in the CRD binds to one nucleotide with specificity determined mainly by aa at position 13.
  • One sample repeat is shown below the protein scheme. Numbers 12/13 refer to aa positions within the repeat. See Jankele et al., Brief Funct Genomics 2014;13:409-19.
  • FIG.2C Repeat types are shown that have specificity for one or several nucleotides. Only bases of the DNA leading strand are shown.
  • FIG.2D Binding of the TALE to GSHS physically sequesters the mobile element enzyme to the same location and promotes transposition to the nearby TTAA sequences. All RVD are preceded by a thymine (T) to bind to the NTR shown in FIG.2A. All of these GSHS regions are in open chromatin and are susceptible to mobile element enzyme activity).
  • FIG.2C also illustrates (bottom) a chimeric mobile element enzyme construct comprising dCas9 linked to one or more guide RNAs.
  • An engineered chimeric mobile element enzyme may include: a guide RNA (gRNA) and an inactivated Cas protein.
  • the gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ⁇ 20 nucleotide spacer that defines the genomic target to be modified.
  • the genomic target of the Cas protein is based upon the sequence present in the gRNA.
  • FIG.3B shows gRNA sequences that physically sequester the mobile element enzyme to GSHS and promotes transposition to the nearby TTAA sequences.
  • FIGs.3A-3E depict examples of construct templates (FIGs.3A-3D) and a system (FIG.3E) comprising a donor DNA and helper RNA.
  • FIG.3A depicts a plasmid construct template that transcribes mobile element enzyme RNA that is later processed with a 5’- m7G cap. Other mobile element enzymes can be substituted.
  • FIG.3B depicts a donor DNA construct template with the transgene VLDLR. Other tissue specific promoters and transgenes can be substituted.
  • FIG. 3C depicts a chimeric mobile element enzyme construct template with a TALE DNA binder. Other TALEs and mobile element enzymes can be substituted.
  • FIG.3D depicts a chimeric mobile element enzyme construct template with a dCas9/gRNA DNA binder. Other dCas9/gRNA and mobile element enzymes can be substituted.
  • FIG.3E depicts a non-limiting example of the system of FIG.1A.
  • the system comprises a nucleic acid (donor DNA), and a nucleic acid (helper RNA) encoding a mobile element enzyme (hyperactive ENGINEERED MOBILE ELEMENT ENZYME).
  • the donor DNA can be any gene of interest (GOI) including those replace, inactive, or provide suicide or helper functions.
  • the GOI can be driven by a predetermined promoter and flanked by insulators to prevent gene silencing.
  • the ITRs are specific for the mammal-derived mobile element enzyme (ENGINEERED MOBILE ELEMENT ENZYME).
  • FIG.3E, panel (B) depicts the helper RNA that is 5’- m7G capped (cap1) with flanking globin 5’- and 3’-UTRs, a 34 polyalanine tail region, and pseudouridine modification.
  • the mobile element enzyme e.g., a hyperactive mobile element enzyme in accordance with the present disclosure
  • FIG. 1A is a non-limiting representation of an AAV production strategy in accordance with embodiments of the present disclosure, using a donor vector to create a HEK293 producer cell line or another cell line including the E1a gene (e.g., engineered CHO-K1 or Vero cell line with E1a) that forms a replication deficient (rep-) AAV particle containing a transgene of interest.
  • E1a gene e.g., engineered CHO-K1 or Vero cell line with E1a
  • the single or dual donor comprising a transgene of interest is incorporated (e.g., by transfection such as electroporation) into a HEK293 cell line or another cell line including the E1a gene.
  • the HEK293 cells are expanded to create a producer cell line and culturing the producer cells creates replication deficient (rep-) AAV particles with the transgene of interest.
  • FIG.1B is a representation of an example of an inducible Rep/Cap and helper AAV donor plasmid construct (Kana r ) used with a helper RNA or DNA to create an AAV producer cell line, in accordance with embodiments of the present disclosure.
  • the donor DNA plasmid of FIG.1B includes helper E2A, E4 and VA genes flanked by insulators and mobile element enzyme recognition ends, to create an AAV producer cell line.
  • FIG.1C is a representation of an example of a nucleic acid (plasmid) encoding a transgene (gene of interest (GOI)) included between AAV inverted terminal repeats (ITRs), in accordance with embodiments of the present disclosure.
  • the plasmids shown in FIGs.1B and 1C can be combined or can be used separately for transfection into an E1+ cell line to produce AAV particle comprising the GOI.
  • FIG.4 depicts a non-limiting representation of a conventional rAAV production system.
  • AAV Rep/Cap genes the ITR-flanked gene of interest (GOI), as well as Ad-helper genes are provided as three separate plasmids, respectively, to a E1a/E1b containing HEK293 cell line or other engineered cell line that contains E1a (e.g., engineered CHO-K1 or Vero cells).
  • the present strategy is to include the AAV rep and cap genes, the AAV vector DNA sequences, and the essential Ad helper genes in a single donor plasmid flanked end sequences recognized by the mobile element enzyme in accordance with embodiments of the present disclosure.
  • the AAV rep and cap genes are included under the control of t-REx, leading to rep and cap gene amplification, the AAV vector DNA sequences, and the essential Ad helper genes in a single donor plasmid flanked by the end sequences recognized by the mobile element enzyme in accordance with embodiments of the present disclosure (FIG.1B).
  • the ability of the integrated AAV helper plasmid to direct the rescue, replication, and packaging of an AAV ITR-flanked transgene (e.g., the transgene of FIG.1C) will be assessed.
  • the integrated AAV helper plasmid can direct the rescue, replication, and packaging of an AAV ITR-flanked transgene.
  • This mobile element enzyme-mediated recombination system is expected to generate an integrated AAV helper plasmid that can facilitate the production of high-titer recombinant AAV particles in a simple two-plasmid transfection protocol.
  • the inducible nature of the rep and cap genes is employed by the use of reagents (e.g., rAAV-5 plasmid reagents) useful for the construction of rAAV-5 vectors bearing other reporter and/or therapeutic transgenes.
  • reagents e.g., rAAV-5 plasmid reagents
  • Example 4 Schematic Illustration for Constructs Used for Establishment of AAV producer stable cell lines, AAV2-H (Plasmid shown in FIG.5), AAV2-HB (Plasmid shown in FIG.6), and AAV-CDH (Plasmid shown in FIG.7) in HEK- 293R-22 tetR-expressing HEK-293 cells.
  • Plasmid with pSF-ITRP5TO1-AAV2H (FIG.5): pSF-ITRP5TO1-AAV2H is an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter with AAV2_REP52/40 ORFs and CAPs (VP1/2/3) under their native P19 or P40 promoter, respectively, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • ITR engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter with AAV2_REP52/40 ORFs and CAPs (VP1/2/3)
  • Plasmid with pSF-ITRP5TO1-AAV2HB (FIG. 6): pSFP5TO1-AAV2HB is an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP78/68 ORFs under the control of P5TO1 promoter and AAV2_REP52/40 ORFs under the native P19 promoter, 2) AAV2 CAPs under the tetO-containing P40 promoter with AAV9 P40 intron, 3) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 4) VA RNAs under the control of HSV-1 ICP4 promoter, and 5) hygro-B resistant gene under the control of TK promoter.
  • Plasmid pSFP5TO1-AAVCDH (FIG.7): pSFP5TO1-AAVCDH is an engineered mobile element enzyme ITR containing plasmid that encodes 1) AAV2_REP 78/68 ORFs under the control of P5TO1 promoter and AAV2_REP52/40 ORFs under the native P19 promoter, 2) Ad5 E2A_IRES_Ad5 E4 ORF6 under the control of the tetO-containing SV40 promoter, 3) VA RNAs under the control of HSV-1 ICP4 promoter, and 4) hygro-B resistant gene under the control of TK promoter.
  • Plasmid pSFA2-EP40CAP2WZ1_SB (FIG.8): This construct encodes AAV2 CAP under the control of the tetO-containing hCMV-derived enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and modified AAV2 P40 intron_1 (a at position 276 substituted for C) and WPRE/modified ICP27 poly A, followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with ITRs for Sleeping Beauty.
  • Plasmid pSFA2-EP40CAP2WZ2_SB (FIG.9): This construct encodes AAV2 CAP under the control of the tetO-containing hCMV enhancer element 3/AAV2 P40 promoter with a modified AAV2 P40 TATA element and modified AAV2 P40 intron_2 and WPRE/modified ICP27 poly A, followed by ICP0 promoter-Zeocin resistant gene-ICP0 poly A cassette flanked with SB ITRs. Establishment of AAV Producer/packaging cell lines a.
  • HEK-293R-22 cells were seeded at about 5 x 10e6 cells per 100 mm dish with normal HEK-293 cells growth medium.
  • Cells were co-transfected with pSF-ITRP5TO1-AAV2H, CMV-Engineered mobile element enzymeB VB200927-4525erk, and pSF-mCMVR at a DNA:Lipofectamine 2000 ratio of 1:2.
  • Transfection medium was removed at 3 and a half hour post transfection followed by addition of normal 293 cells growth medium.
  • Transfected cells were passed on day 3 post transfection and grew in 293 cells growth medium in the presence of hygro-B at a concentration of 100 ug/ml.
  • Hygro-B resistant single stable AAV-H colonies were then selected and analyzed by 1) ddPCR analyses with REP, E2A, ORF6, VA RNA and hygro-B resistant gene-specific primers, 2) western blot analysis with anti-AAV-2 REPs and CAPs specific antibodies, and 3) their ability to support AAV genome amplification following transduction with AAV2-EGFP viral particles.
  • HEK-293R-22 cells were seeded at about 5 x 10e6 cells per 100 mm dish with normal HEK- 293 cells growth medium.
  • Cells were co-transfected with VB220909-1308jwt_pSF-ITRP5TO1-AAV2HB, CMV- Engineered mobile element enzymeB VB200927-4525erk, and pSF-mCMVR at a DNA:Lipofectamine 2000 ratio of 1:2.
  • Transfection medium was removed at 3 and a half hour post transfection followed by addition of normal 293 cells growth medium.
  • Transfected cells were passed on day 2 post transfection and grew in 293 cells growth medium in the presence of hygro-B at a concentration of 100 ug/ml.
  • Hygro-B resistant single stable AAV-HB colonies were then selected and analyzed by 1) ddPCR analyses with REP, E2A, ORF6, VA RNA and hygro-B resistant gene-specific primers, 2) western blot analysis with anti-AAV-2 REPs and CAPs specific antibodies, and 3) their ability to support AAV genome amplification following transduction with AAV2-EGFP viral particles.
  • AAV serotype adaptable (Cap minus or Cap-) stable cell lines AAV-CDH (Plasmid shown in FIG.7) cells
  • HEK-293R-22 cells were seeded at about 5 x 10e6 cells per 100 mm dish with normal HEK-293 cells growth medium.
  • Cells were co-transfected with pSF-ITRP5TO1- AAVCDH, CMV-Engineered mobile element enzymeB VB200927-4525erk, and pSF-mCMVR at a DNA:Lipofectamine 2000 ratio of 1:2. Transfection medium was removed at 3 and a half hour post transfection followed by addition of normal 293 cells growth medium.
  • Transfected cells were passed on day 3 post transfection and grew in 293 cells growth medium in the presence of hygro-B at a concentration of 100 ug/ml or 50 ug/ml.
  • Hygro-B resistant single stable AAV-CDH Plasmid shown in FIG.7 colonies were then selected and analyzed by 1) ddPCR analyses with REP, E2A, ORF6, VA RNA and hygro-B resistant gene-specific primers, 2) western blot analysis with anti-AAV-2 REPs specific antibody, and 3) their ability to support AAV genome amplification following transduction with AAV2-EGFP viral particles. d.
  • AAV2-capsid minus stable lines Clone CDH-48, CDH-24 and CDH-129 cells HEK-293 were seeded at about 4 to 5.5 x 10e6 cells per 100 mm dish with normal HEK-293 cells growth medium.
  • Cells were co-transfected with of pSFA2-EP40CAP2WZ1-SB, pSFA2-EP40CAP2WZ2-SB, pSF-mCMVR, and sleeping beauty transposase SB100X Helper mRNA (VB210111- 1032qxg) at a DNA:Lipofectamine 2000 ratio of 1:2. Transfection medium was removed at 3 and a half hour post transfection followed by addition of normal 293 cells growth medium. Transfected cells were passed on day 2 post transfection with normal HEK-293 cells growth medium. Cells were then grown in HEK-293 cells growth medium in the presence of 100 ug/ml of zeocin.
  • Zeocin resistant single stable colonies were then selected and analyzed by 1) ddPCR analyses with REP, Cap, E2A, ORF6, VA RNA and zeocin resistant gene-specific primers, 2) western blot analysis with anti-AAV-2 REPs and CAPs specific antibodies, and 3) their ability to support AAV genome amplification following transduction with AAV2-EGFP viral particles.
  • ddPCR analyses for an engineered mobile element enzyme mediated Transposon Integration, AAV Genome Amplification/Rep Function, Cap Copy Insertion via Sleeping Beauty, and physical titer of rAAV particles.
  • AAV genome amplification is supported in either AAV2 producer cell line clone H-17 (FIG.10A – 10B, Cap+) or AAV serotype adaptable packaging cell line clone CDH-13 (FIG.11A – 11B, Cap-).
  • AAV genome amplification was used as a reference for expression of functional AAV Rep proteins in the AAV-H Clone H-17 or AAV-CDH (Plasmid shown in FIG.7) clone CDH-13 cells.
  • Commercially available rAAV2-EGFP particles from SignaGen at 75 VGC/cell was used to transduce 3 x 10e6 cells/dish of Clone H-17, CDH-13, or HEK-293 (AAV Rep- control) cells.
  • GFP images (FIG.10A – FIG.11B) were taken ⁇ 120 hours post-transduction using Zeiss AxioVert A1 microscope and Axiocam 705 mono camera, followed by cell harvesting, genomic DNA extraction, and ddPCR analyses for quantification of the rAAV2-EGFP genome copy number per nanogram (ng) of total genomic DNA.
  • ddPCR results at right The results clearly indicated that the rAAV-EGFP replicated in doxycycline dose responsive manner (ddPCR results at right), suggesting the inducible expression of functional AAV Rep proteins in the Clone H-17 and CDH-13 cells.
  • FIG.12A shows the results of an western blot analysis illustrating cells from selected AAV2-HB (Plasmid shown in FIG. 6) cell clones HB-36, HB-42, HB-118, and HB-120 were treated with10ng/ml doxycycline for 72 hours followed by extraction with RIPA buffer in the presence of proteinase inhibitor. The resulting cell lysate supernatants were subjected to electrophoresis and western blot analyses.
  • FIG.12B shows the results of a ddPCR analysis for AAV Genome Amplification / Rep function Assay.
  • the AAV-HB stable clone #36, 42, 118 and 120 cells were transduced with the rAAV2-EGFP particles from SignaGen at low MOI, and genomic DNA extracted/ddPCR performed for quantification of the rAAV2-GFP genome copy number per nanogram (ng) of total genomic DNA analyzed.
  • AAV2 full producer stable cell pools CDH-24/Cap-SB, CDH-48/Cap-SB, and CDH-129/Cap-SB with sleeping beauty transposase-mediated integration of AAV2 CAPs-expressing cassettes into AAV-CDH (Plasmid shown in FIG.7) serotype adaptable (Cap minus or Cap-) stable cell lines CDH-24, CDH48, and CDH- 129.
  • FIG.14A shows ddPCR analyses of SB-mediated AAV2 Cap transposon integration in each Zeocin-selected stable pools. Average Cap copy number integration in each pool is summarized at the bottle of the penel.
  • FIG.14B shows western blot analysis of SB-mediated AAV2 Cap transposon integration in each Zeocin-selected stable pools.
  • CDH-48/Cap-SB stable pool increased with doxycycline dosages and over time.
  • Cells from CDH-48/Cap-SB stable pools were incubated either in the presence of 0, 5, 10, 25, 50, or 100ng/ml doxycycline and harvest at 72 hours post induction; or incubated with 50ng/ml doxycycline and harvested at 24, 48, 72, or 96 hours post induction (FIG.16).
  • Cell extraction with RIPA buffer containing proteinase inhibitors, western blot, and ddPCR analyses of the CDH-48/Cap-SB (AAV2-capsid minus stable lines Clone CDH-48) samples were caried out as described previously.
  • the results show that expression of AAV2 Cap and Rep proteins increased over time in the presence of 50ng/ml doxycycline.
  • the Cap and Rep expression also increased in response to increasing amounts of doxycycline.
  • the cells from the CDH-48/Cap-SB stable pools were transduced with the rAAV2-EGFP particles from SignaGen at 500 VGC/cell in the presence or absence of 50ng/ml. 4 days post-transduction, culture media were removed, and cells harvested and washed twice with fresh growth media to remove the residue doxycycline.
  • rAAV-EGFP containing supernatants were collected following centrifugation at 2500rpm for 10 minutes AAV physical titer in each sample was measured by ddPCR as described above and data presented in FIG.17A.
  • the unconcentrated supernatants were used for transduction of CDH-48 Cap- cells at 1.5ml/well of the 6-well tissue culture plates in duplicates.
  • the transduction media were replaced next day with fresh growth media and cells were harvested after additional 2 days incubation followed by fgenomic DNA extraction.
  • ddPCR analyses of the genomic DNA samples were performed to evaluate copies of the rAAV-GFP copies introduced into CHD-48 Cap- cells through transduction and results presented in the FIG.17B.
  • the ddPCR results from both panels indicated that rAAV-EGFP particles were produced in response to doxycycline induction and the produced rAAV particles were competent for transduction. Characterization of Zeocin-resistant single clones selected from the CDH-48/Cap-SB stable pool.
  • Zeocin resistant single stable colonies were selected from the CDH-48/Cap-SB stable pool and analyzed by 1) ddPCR analysis for SB-mediated AAV2-Cap cassette integration (FIG.18A); 2) western blot analysis with antibodies specific for AAV2 CAP (FIG.18B), and AAV2 Rep (FIG.19A); and 3) ddPCR assays to evaluate their ability to support AAV genome amplification following transduction with low VGC/cell of AAV2-EGFP viral particles (FIG.19B).
  • FIG.18A indicated that SB-mediated AAV2-cap cassette integration ranged from 20-90 copies per ng of total genomic DNA.
  • Transposon copy number ddPCR The Transposon Copy Number ddPCR Assay quantifies the copy numbers of five elements in the Engineered mobile element enzyme transposon per genome of HEK293R-22 based cells. These elements are AAV2 Rep, AAV2 Cap, Ad5 E2A, Ad5 E4 Orf6, and VA RNA. Genomic DNA was extracted from samples with the Zymo Research Quick-DNA MiniPrep Kit (D3025) according to the manufacturer’s protocol.
  • Sample DNA concentrations were quantified by the Thermo Qubit 1X dsDNA Broad Range Assay Kit (Q33266) according to the manufacturer’s protocols and normalized to the same concentration in TE.
  • ddPCR reactions were assembled with Manufacturer’s Supermix for Probes (no dUTP), a FAM-labeled hydrolysis probe-based ddPCR assays for one of the transposon elements, a HEX-labeled hydrolysis probe-based ddPCR assay for RPP30, HindIII restriction enzyme, and water according to Manufacturer’s ddPCR protocol. Sample DNA was added to reaction wells at inputs of 4 ng and 20 ng per well to ensure that at least one reaction per sample was within the range of the assay.
  • AAV Genome Amplification / Rep function Assay quantifies the copy number of AAV-GFP genomes per nanogram (ng) of genomic DNA analyzed. Genomic DNA was extracted from cells with the Zymo Research Quick-DNA MiniPrep Kit (D3025) according to the manufacturer’s protocol. Sample DNA concentrations were quantified by the Thermo Qubit 1X dsDNA Broad Range Assay Kit (Q33266) according to the manufacturer’s protocols and normalized to the same concentration in TE.
  • ddPCR reactions were assembled with Manufacturer’s Supermix for Probes (no dUTP), a FAM-labeled hydrolysis probe-based ddPCR assay for EGFP, HindIII restriction enzyme, and water according to Manufacturer’s ddPCR protocol.
  • Sample DNA was added to reaction wells at inputs of 0.02 to 20 ng per well to ensure that at least one reaction per sample was within the range of the assay.
  • Droplets were generated by the automated QX200 AutoDG system and thermal cycled according to Manufacturer’s recommended parameters, with an annealing Tm of 60 ⁇ . Thermal cycled droplets were analyzed by Direct Quantitation on the QX200 Droplet Reader. Sample data was inspected for sufficient droplets (>10,000) and properly identified positive and negative droplets prior to analysis.
  • the QX200 droplet reader software automatically calculates the target (EGFP) copies per uL of reaction volume.
  • c Cap Copy Number ddPCR Assay: The Cap Copy Number ddPCR Assay quantifies the copy number of AAV2 Cap genes per genome of HEK293-based cells. Genomic DNA was extracted from samples with the Zymo Research Quick-DNA MiniPrep Kit (D3025) according to the manufacturer’s protocol.
  • Sample DNA concentrations were quantified by the Thermo Qubit 1X dsDNA Broad Range Assay Kit (Q33266) according to the manufacturer’s protocols and normalized to the same concentration in TE.
  • ddPCR reactions were assembled with Manufacturer’s Supermix for Probes (no dUTP), a FAM-labeled hydrolysis probe-based ddPCR assay for AAV2 Cap, a HEX-labeled hydrolysis probe-based ddPCR assay for RPP30, HindIII restriction enzyme, and water according to Manufacturer’s ddPCR protocol. Sample DNA was added to reaction wells at inputs of 4 ng and 20 ng per well to ensure that at least one reaction per sample was within the range of the assay.
  • AAV physical titer ddPCR quantifies the copy number of AAV-GFP genomes per mL of AAV sample analyzed.
  • a series of eight 10-fold serial dilutions (10E0-10E-7) of AAV samples were created to ensure that several dilutions would fall within the range of ddPCR quantitation.
  • Serial dilutions were created in nuclease-free water with 0.05% Pluronic F-68 (Gibco 24040-032).
  • AAV particles were lysed by incubation at 95 ⁇ for 10 minutes, followed by cooling to 4 ⁇ at a rate of 3 ⁇ per minute.
  • ddPCR reactions were assembled with Manufacturer ⁇ s Supermix for Probes (no dUTP), a FAM-labeled hydrolysis probe-based ddPCR assay for EGFP, HindIII restriction enzyme, and water according to Manufacturer ⁇ s ddPCR protocol. AAV sample dilutions were added to reaction wells at 5 uL per well. Droplets were generated by the automated QX200 AutoDG system and thermal cycled according to Manufacturer ⁇ s recommended parameters, with an annealing Tm of 60 ⁇ . Thermal cycled droplets were analyzed by Direct Quantitation on the QX200 Droplet Reader. Sample data was inspected for sufficient droplets (>10,000) and properly identified positive and negative droplets prior to analysis.
  • the QX200 droplet reader software automatically calculates the target (EGFP) copies per uL of reaction volume.
  • Western Blot analyses for detecting AAV Rep and Cap Expression a.
  • Cell lysate preparation Upon wash and scrape, ⁇ 1+E7 cells in 10 ml of same PBS containing 1mM PMSF and 100ug/ml TPCK or HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) (Cat# 78441) were pelleted down by centrifugation at 3000 rpm for 15 min at 4c and resuspend in 600 ul of RIPA buffer containing 1mM PMSF, 100ug/ml TPCK, and 50ug/ml leupeptin or Pierce Protease and Phosphatase Inhibitor Mini Tablets (cat# A32959) .
  • AAV Rep, AAV Cap, and host cell Cyclophilin B (a loading control) on PVDF membranes were probed with mouse anti-AAV2 Rep (clone 303.9, Progen Catlog# 61069 at 1:100x), mouse anti-AAV VP1/VP2/VP3 (clone B1, Progen 65158, supernatant at 1:25x), or mouse anti-Cyclophilin B (Abcam, ab236760 at 1:10,000x) followed by HRP conjugated secondary goat-anti-mouse IgG H&L antibody (AbCam, ab6789 at 2000x to 10,000x).

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Abstract

L'invention propose un procédé de fabrication d'une lignée cellulaire productrice et d'encapsulation de particule virale. La lignée cellulaire est basée sur un vecteur de virus adéno-associé (AAV), la lignée cellulaire est amenée à exprimer, en association avec la particule virale, un transgène souhaité.
PCT/US2023/078957 2022-11-07 2023-11-07 Production de virus adéno-associé (aav) Ceased WO2024102739A2 (fr)

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WO2026050618A1 (fr) * 2024-08-29 2026-03-05 Regeneron Pharmaceuticals, Inc. Constructions polynucléotidiques et lignées cellulaires de production d'aav recombinants

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GB201816919D0 (en) * 2018-10-17 2018-11-28 Glaxosmithkline Ip Dev Ltd Adeno-associated viral vector producer cell lines
US12054738B2 (en) * 2020-07-30 2024-08-06 Shape Therapeutics Inc. Stable cell lines for inducible production of rAAV virions
WO2023173105A2 (fr) * 2022-03-10 2023-09-14 Saliogen Therapeutics, Inc. Système inducteur/répresseur de tétracycline et production de particules virales simplifiée

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WO2026050618A1 (fr) * 2024-08-29 2026-03-05 Regeneron Pharmaceuticals, Inc. Constructions polynucléotidiques et lignées cellulaires de production d'aav recombinants

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